PLGA-HAp Composite Scaffold for Osteochondral

Subscriber access provided by EKU Libraries

Tissue Engineering and Regenerative Medicine

Bilayered PLGA/PLGA-HAp Composite Scaffold for Osteochondral Tissue Engineering and Tissue Regeneration Xiangyu Liang, Pingguo Duan, Jingming Gao, Runsheng Guo, Zehua Qu, Xiaofeng Li, Yao He, Haoqun Yao, and Jiandong Ding ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00552 • Publication Date (Web): 28 Aug 2018 Downloaded from http://pubs.acs.org on August 31, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Biomaterials Science & Engineering

Bilayered PLGA/PLGA-HAp Composite Scaffold for Osteochondral Tissue Engineering and Tissue Regeneration

Xiangyu Liang1,†, Pingguo Duan2,†, Jingming Gao1, Runsheng Guo2, Zehua Qu1, Xiaofeng Li2, Yao He1, Haoqun Yao2, Jiandong Ding1,* 1

State Key Laboratory of Molecular Engineering of Polymers, Department of Macromolecular Science, Fudan University, Shanghai 200438, China.

2

Department of Orthopaedic Surgery, The First Affiliated Hospital of Nanchang University, Nanchang 330006, China.

*

Correspondence address. Tel.: +86-21-31243506. E-mail: [email protected].

†

These authors contributed equally to this work.

1

ACS Paragon Plus Environment

ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT: This study is aimed at investigation of the osteochondral regeneration potential of bilayered PLGA/PLGA-HAp composite scaffolds with one layer made of biodegradable polymer poly(D,L-lactide-co-glycolide) (PLGA) and another layer made of PLGA polymeric matrix coated by bioactive ceramics hydroxyapatite (HAp). The composite scaffolds were fabricated by compression molding/particle leaching and plasma-treated surface deposition. The pore morphology, mechanical properties and surface deposition of the scaffold were characterized, and the growth of bone marrow derived mesenchymal stem cells or medicinal signaling cells (MSCs) in the scaffold was verified. Thereafter, rabbit models with an artificial osteochondral defect in joint were randomized into three treatment groups: virgin bilayered scaffold, bilayered scaffold pre-seeded in vitro with MSCs, and untreated blank control. At a 16-week postoperation, both the virgin scaffolds and cell-seeded bilayered scaffolds exhibited osteochondral repair, as verified by biomechanics analysis, histological evaluations and Western blot etc. The results highlighted the potentiality of the bilayered PLGA/PLGA-HAp composite scaffold for osteochondral tissue engineering, and in particular tissue regeneration or in situ tissue induction, probably by recruiting the local cells towards chondrogenic and osteogenic differentiation in the porous biomaterials.

KEYWORDS: Bilayered scaffold; biodegradable polymers; composite materials; tissue regeneration; osteochondral repair

2


Page 2 of 45

Page 3 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


INTRODUCTION Articular cartilage lesions are a common and increasingly serious problem due to disease, sports and progressive aging, and the lesions can cause persistent symptoms of joint pain and constrained motion of the knee joints.1 Once damaged, an articular cartilage is hard to self heal due to no blood supply and limited chondrocyte migration constrained by the rich extracellular matrix (ECM) in this tissue. The repair of an articular cartilage defect has thus been an intractable challenge in the field of regenerative medicine.2-3 In order to treat the articular cartilage lesions, one has developed arthroscopic microfracture,

arthroplasty,

osteotomy,

autologous

chondrocyte

implantation

and

conservative treatments.4 These techniques are limited to defect size, palliative or inducing donor-site morbidity.5 Tissue engineering emerges as a promising alternative strategy for cartilage regeneration by implanting porous scaffolds to provide a three-dimensional support for cell growth and in situ tissue regeneration.2, 6-9 However, repair of cartilage merely by a single-layer scaffold probably brings about the problem of poor integration between cartilage and subchondral bone. It has been reported that articular cartilage cannot be independently repaired without assistance from subchondral bone.10 The subchondral bone plays, as a strong support of cartilage, an important role on stress transmission in the knee joints.11 Moreover, articular cartilage and subchondral bone are two tissues with different moduli, components, physiological structures and physical functions, indicating that a single-layer scaffold cannot fully meet the requirements of osteochondral repair.12-13 Hence, the rehabilitation of the cartilage layer or chondral layer might be carried out simultaneously with the reconstruction of the subchondral bone layer or bony layer. Some bilayered scaffolds have been proposed to mimic the cartilage layer and subchondral bone layer.11, 14-18 For instance, Yin group reported a bilayered poly(L-glutamic acid)/chitosan scaffold seeded with adipose stem cells, which was implanted into rabbit models to regenerate the hyaline-like cartilage demonstrated by the histological examination, biomechanical and biochemical analysis.19 TruFit™ (Smith & Nephew, USA) is made of semiporous poly(D,L-lactide-co-glycolide) (PLGA), calcium sulfate, and polyglycolide (PGA) fibers, yet showed a controversial effectiveness for osteochondral20-22; Agili-C™ (CartiHeal Ltd, Israel) is a crystalline coral aragonite/hyaluronic acid bilayer scaffold, showing the 3



restoration of the osteochondral defect23. A bilayered scaffold seeded with cells before implanting into osteochondral defect has a positive efficacy of repair, because cells seeded on the scaffolds can be triggered to differentiate by local in vivo microenvironment24-25. A critical question is then raised about the necessity of cell seeding for an osteochondral bilayered scaffold, or whether a virgin bilayered scaffold alone could have an equiponderant osteochondral regeneration or not. Cell-loaded scaffolds have significant restrictions on long-time preparation, complicated examination and expensive regulatory approval of applications. Food and drug administration (FDA) might consider a bilayered scaffold as a “medical device”, while a scaffold-cell mixture could, so far, only be regarded as a biological technique instead of a product. These regulatory requirements thereby enhance much interest in cell-free bilayered scaffolds for osteochondral repair. So, it is pretty necessary even emergent to make it clear whether or not a merely virgin scaffold can be used for an in situ osteochondral induction or regeneration. An appropriate composite scaffold might lead to a very good repair efficacy even without introduction of any seed cells, as schematically showed in Figure 1a. The present report is aimed to fabricate a bilayered composite scaffold to check such a hypothesis as an acellular strategy.

4


Page 4 of 45

Page 5 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1. Repair of an osteochondral defect in a rabbit knee joint by a PLGA/PLGA-HAp bilayered scaffold. (a) Schematic presentation of the efficacy. “Blank control” refers to the group with the defect untreated, “Scaffold” indicates the group of implanting just a porous scaffold to examine the effect of in situ tissue regeneration, and “Scaffold with MSC” indicates the group of implanting both a porous scaffold and external seed cells. The dashed line presents the efficacy of the normal tissue. (b) Schematic presentation of the site of the defect and implant. (c) Matrix material and the fabrication of the composite bilayered porous scaffold. The cartilage layer scaffold (PLGA) and subchondral layer scaffold (PLGA/HAp) were prepared separately, and then glued by dichloromethane (CH2Cl2) to obtain a bilayered scaffold.

Our group had ever prepared bilayered scaffolds composed of merely PLGA polymers.26-27 According to our previous studies26-27, the scaffold with a porosity of 92% in the cartilage layer and a porosity of 77% in the subchondral bone layer exhibited the best efficacy in tissue engineering. And pore sizes of 100 - 200 µm in the cartilage layer and 300 450 µm in the subchondral bone layer generated the best results. These findings of bilayered 5



scaffold design parameters are very helpful for subsequent research for osteochondral tissue engineering and regenerative medicine. In light of biomimetics, the merely polymeric scaffold might not be ideal for the subchondral bone. Our previous study of tissue engineering of segmental bone indicated that introduction of some bioceramics such as hydroxyapatite (HAp) was much helpful for promoting osteogenesis.28 In this work, we fabricated bilayered PLGA/PLGA-HAp scaffolds and implanted them into the femoral condyles of rabbits (Figure 1b) to evaluate the physicochemical properties and biological performance of the composite scaffolds. The bilayered scaffold was fabricated by gluing the cartilage layer scaffold (PLGA) and subchondral layer scaffold (PLGA/HAp) as showed in Figure 1c. Aliphatic polyester is a class of popular biodegradable polymers,14, 29-37 among which PLGA is very important as a matrix for a tissue engineering scaffold as its high adjustability of degradation rate.38 Sodium chloride particles are employed as porogen to fabricate the cartilage layer and subchondral bone layer of PLGA porous scaffolds by room-temperature compression molding and porogen leaching.27,

39

We also examined the effect of tissue

engineering by implanting porous scaffolds and seed cells. Mesenchymal stem cells or medicinal signaling cells40-41 (MSCs) derived from bone marrow were employed as a model cell type, which has been an important type of seed cells in tissue engineering.42-47 It is well known that HAp is one of the most important elements of bone and plays an outstanding role on biomechanics and osteogenic differentiation of MSCs.48-51 In addition, HAp would buffer the acidic products of PLGA degradation and may thereby contribute to avoid an unfavorable environment for the cells and tissue owing to a decreased pH.52-53 So, we deposited the HAp on the interior surfaces by immersing them into modified simulated body fluids (SBF) so as to enhance the biocompatibility and mechanical property of the subchondral bone layer scaffolds. We implanted as-fabricated bilayered scaffolds alone and also the scaffolds with MSCs into osteochondral defects in rabbit knee joints (Figure 1b) to assess their safety and regenerative potential of the virgin bilayered scaffold on osteochondral repair in vivo. Besides in vitro examination of cell growth in the porous scaffold and global observation of in vivo tissue repair, we also investigated the regenerated cartilage and subchondral bone with 6


Page 6 of 45

Page 7 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


micro-CT observations, histological analysis, biomechanical tests based on atomic force microscopy (AFM), and Western blot of relevant protein expression. Our work might be of its impact for biomaterial design and potential clinical application of composite scaffolds.

MATERIALS AND METHODS Ethics Statement. All animal experiments in this work were carried out in strict accordance with the recommendation in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health54. The protocol was approved by the Committee on the Ethics of Animal Experiments of The First Affiliated Hospital of Nanchang University. All surgeries were performed under the anesthesia with 3% pentobarbital sodium (1 mL/kg), and all efforts were made to minimize their suffering. Fabrication of PLGA Scaffolds. Commercial PLGA with an 85:15 molar ratio of lactide/glycolide (LA/GA) was purchased from Purac Biochem (Gorinchem, The Netherlands), and inherent viscosity was 2.93 dL/g. The porous PLGA scaffolds were fabricated by room-temperature compression-molding/porogen-leaching as described previously.39,

55-57

Briefly, PLGA granules were dissolved in dichloromethane (10%

weight/volume), and sodium chloride (NaCl) particles with the sieved size ranges of 100 200 µm (cartilage layer) and 300 - 450 µm (subchondral bone layer) were used as porogens. Porosities and pore sizes of the cartilage layer and subchondral bone layer scaffolds were controlled by porogen fractions and sizes, respectively. The paste-like mixture was pressed into a mold with a predesigned size and kept molding under 24-hour pressure. Cylindrical samples were obtained after releasing the mould and laid in the fume hood for further solvent evaporation for another 24 h. After leaching porogen by deionized water, two kinds of PLGA scaffolds with different porosities and pore sizes were obtained as the cartilage layer and subchondral bone layer. Fabrication of PLGA/HAp Scaffolds. Oxygen plasma pretreatment (DT-03, Suzhou OPS Plasma Technology Co., Ltd., Jiangsu, China) was employed to improve the interior surface hydrophilicity and ion anchorage of subchondral-bone-layer porous scaffolds. The chamber was vacuumized to 8 Pa, then feeding oxygen at a flow rate of 50 standard-state cubic centimeter per minute (sccm). After the chamber pressure was stabilized, plasma 7



Page 8 of 45

treatment was carried out at 50 W power for 8 min. Prior to being taken out, the treated scaffolds were exposed to an oxygen atmosphere in the chamber for another 5 min. After the oxygen plasma pretreatment, the as-prepared scaffolds were immediately immersed into the modified SBF as described previously28, 58-59, and vacuumed to ensure that interior pores were fully filled with the modified SBF. The immersion was kept at room temperature for 12 h. Thereafter, incubation was always kept in a water bath at 37°C. We added NaHCO3 into the modified SBF to adjust pH 6 - 7 to trigger depositing. The incubation was kept for 9 h in order to fully deposite the HAp, and the modified SBF was exchanged every 3 h for holding the concentration of the effective ions. The last step is to rinse the deposited porous scaffolds with Milli-Q water, and kept them in a vacuum dryer for subsequent use. Fabrication of Bilayered PLGA/PLGA-HAp Scaffolds. As-prepared cartilage layer scaffold (PLGA) and subchondral layer scaffold (PLGA/HAp) were glued to a bilayered PLGA/PLGA-HAp scaffold by coating CH2Cl2 on the contact surface of them. Then resultant bilayered scaffolds were cut into 4 mm in diameter and 5 mm in height (cartilage layer: 1 mm, subchondral layer: 4 mm). Porosity Measurements of the Cartilage-layer Scaffold and Subchondral-layer Scaffold. The porosity of as-prepared cartilage-layer scaffold (PLGA) and the preform of the subchondral-layer scaffold (PLGA) were measured by a liquid replacement method60. Briefly, anhydrous ethanol (AR 99.7%, Shanghai Titan Scientific Co., Ltd.) was employed as the liquid to measure the volume of the pores within the scaffold. A dry porous scaffold was weighed as mscaffold. Anhydrous alcohol was completely penetrated into pores of scaffolds by vacuumized. Then we immediately put it into a weighing bottle full of anhydrous alcohol, which was weighed as m1. After the porous scaffold was removed, we recorded the mass of the weighing bottle with residual anhydrous alcohol as m2. Porosity was then calculated by following formula:

ϕ=

(m1 - m2 - mscaffold ) / ρethanol × 100% (m1 - m2 - mscaffold ) / ρethanol + mscaffold / ρPLGA

(1)

Here, ρethanol and ρPLGA denote the density of anhydrous alcohol and that of PLGA, respectively. 8


Page 9 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


For the subchondral-layer scaffold (PLGA/HAp), the theoretical porosity was calculated by an amendment method as described previously28. Briefly, the mass of HAp deposition was determined by subsequent thermogravimetric analysis detection, and then the weight fraction of the inorganic component fw, HAp was obtained. The theoretical porosity was calculated by

ϕ=

(m1 - m2 - mscaffold ) / ρ ethanol × 100% (m1 - m2 - mscaffold ) / ρ ethanol + (1 − fw, HAp ) mscaffold / ρ PLGA (2)

+ fw, HAp mscaffold / ρ HAp Here, ρHAp denotes the density of the bioceramic.

TGA Analysis of the Content of the HAp Deposition on the Interior Surfaces of Subchondral-layer PLGA Scaffold. Thermogravimetric analysis (TGA, PE, Pyris 1, USA) was carried out to detect the mass of HAp deposition on the interior surfaces of the subchondral-layer PLGA scaffold under a nitrogen atmosphere at a flow rate of 40 mL/min. We examined the weight from 50°C to 800°C at a heating rate of 20°C/min. FESEM-EDS Analysis of As-fabricated Bilayered Scaffolds. Field-emission scanning electronic microscopy (FESEM, Zeiss, Ultra 55, Germany) was carried out to observe the morphology of interior pores of bilayered PLGA/PLGA-HAp scaffold slices. And energy dispersive X-ray spectroscopy (EDS, Zeiss, Germany) was employed to analyze the calcium-phosphorus ratio of the deposition on the interior surfaces of subchondral-layer PLGA scaffold. Before the FESEM-EDS testing, a thin layer of gold was sputtered on the sample surface for 60 s. The heater voltage in the microscope was set at 20 kV. XRD Analysis of the Deposition on the Interior Surfaces of Subchondral-layer PLGA Scaffold. X-ray diffraction (XRD, X’Pert Pro, PANalytical B.V., the Netherlands) was employed to qualitatively characterize the deposition on the interior surfaces of subchondral-layer PLGA scaffold. We used commercial hydroxyapatite as standard. Compression Tests to Determine Mechanical Properties of the Bilayered Scaffolds at Both Dry and Wet States. The compressive properties of the cartilage-layer, subchondral-layer and bilayered scaffolds were characterized by measuring the stress-strain curves with the approach described in our previous work.26-27, 61 Cylindrical porous scaffolds with a diameter of 4 mm and a height of 5 mm were used as testing specimens at both “dry” 9



Page 10 of 45

and “wet” states. The “dry-state” testing was conducted in a “dry” state at room temperature. For the “wet-state” tests, the specimens were pre-wetted with ethanol first and then with phosphate buffered saline (PBS) solution to replace the ethanol via a cyclic vacuumization and air-charge method39, 62 to make sure that the specimens were fully permeated with PBS solution until completely submerged into the PBS medium. After 24-hour immersion into PBS, the mechanical testing of the specimens at both “dry” and “wet” states were carried out by using an electromechanical universal testing system (5966, Instron, USA) at a speed of 6.0 mm/min till 80% strain or facture under a constant temperature and humidity condition. After the mechanical testing, normalization processing was used to compare the moduli of “dry-state” and “wet-state” scaffolds. Firstly, the mean value of the each group of “dry-state” modulus was regarded as each reference value (100%). Then all the relative values of the raw “dry-state” and “wet-state” moduli of each group at different scaffolds were gathered as a set, respectively. Eventually, two sets of “dry-state” and “wet-state” moduli were compared. MSC Seeding onto Bilayered PLGA Scaffold and In Vitro Culture. According to the previous protocol63-67, MSCs were isolated from the bone marrow of 7-day old neonatal Sprague-Dawley (SD) rats purchased from Shanghai Laboratory Animal Research Center (China). Before cell seeding, each scaffold slice of 4 mm in diameter and 2 mm in height were sterilized by soaking into 75% ethanol for 24 h, washed in phosphate buffered saline (PBS, pH 7.2 - 7.6, Gibco) three times and then in Dulbecco’s modified Eagle medium (DMEM, Hyclone, Logan, UT, USA) for another three times. As-cultured MSCs in complete medium at a concentration of 5 × 107 cells per mL were injected into the chondral layer of the bilayered scaffold by using a 1-cc syringe as showed in ®

Figure 1c. The scaffolds with seeding cells were placed into 24-well culture plates (Costar , Corning, USA) and incubated at 37°C supplemented with 5% CO2 and 95% humidity for 2 h to make cells adhere well to the scaffold, and incubated for 7 days with the medium exchanged every 48 h before being used in the next experiment. The untreated scaffolds were incubated under the same condition, which served as the control. Observation of MSC Growth in Porous Scaffolds. We observed the MSCs adhering on the interior surface of scaffolds with an SEM microscope (VEGA 3 XMU, TESCAN, Czech). After 7-day culture, the cell-seeded scaffolds were gently fixed with 4% 10


Page 11 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


paraformaldehyde at room temperature for 1 h. Then dehydration of gradient ethanol was carried out. Gold was sputtered onto the surfaces of samples for 60 s, and adherent MSCs were observed by SEM. We also used an optical microscope to observe cells on porous scaffolds after 7-day culture. Prior to the observations, filamentous actins (F-actins) and nuclei of MSCs on the scaffolds were fluorescently stained by phalloidin-tetramethyl rhodamine B isothiocyanate (phalloidin-TRITC) and 4',6-diamidino-2-phenylindole (DAPI). Briefly, MSCs on the scaffolds were thoroughly rinsed with PBS, fixed into 4% paraformaldehyde at room temperature for 10 min and rinsed with PBS for 5 min three times, then treated by 0.1% Triton X-100 for 10 min for membrane permeation and rinsed with PBS for 5 min three times again. Firstly, in order to stain F-actins, MSCs were incubated with 1 mg/mL phalloidin-TRITC (Sigma) at room temperature in dark for 1 h, and then rinsed with PBS for 5 min three times. Afterwards, the nuclei of MSCs were stained. We added 5 mg/mL DAPI (Sigma) at room temperature in dark for 10 min, and the sample was thoroughly rinsed with Milli-Q water for several times. An inverted fluorescence microscope (Axiovert 200, Zeiss, Germany) was employed to observe the immunofluorescence of stained MSCs. The fluorescence micrographs were captured by a digital CCD camera (AxioCam HRC, Zeiss, Germany).68 Surgical Implantation. Thirty-six skeletal mature New Zealand white rabbits (5-6 months old, randomly selective gender) with weight of 2.9 - 3.3 kg were enrolled in the study. After a one-week acclimation, the rabbits were anaesthetized with pentobarbital sodium (1 mL/kg) and fixed the limbs up. The knee joints were disinfected after being shaved. Then a medial parapatellar incision was made on the bilateral knee joints until the femoral condyle was exposed. A surgical drill bit (customized) with scale marks was employed to create an osteochondral defect (4 mm in diameter and 5 mm in depth) centered on the femoral medial condyle, and the defect was flushed with sterile normal saline. Thereafter, the cell-seeded and virgin bilayered PLGA/PLGA-HAp scaffolds were press-fitted into the medial condyle in each knee joint. The cell-seeded and virgin bilayered PLGA/PLGA-HAp scaffolds were set up as one implanted pair. Rabbit knee joints were assigned randomly to the implants of two pairs in 24 11



rabbits (8 and 16 weeks, n = 6 for each scaffold group). Besides, the untreated lateral condyle as a normal group, and the medial condyle defect in the same knee joints remained blank as a blank control group. After implantation, the surgical incision like joint capsule and skin were sutured layer by layer. All rabbits were fed in cages with tap water and food ad libitum and allowed to move freely. Last but not least, gentamycin (4 mg/kg) was injected intramuscularly for 3 days after surgical implantation and once a day to avoid postoperative infects. Micro-CT Observations of the Osteochondral Defects and Efficacies of Repair of Rabbit Knee Joints. A high-resolution micro-CT imaging system (Bruker SkyScan 1176, USA) was employed to observe the osteochondral defects and assess the repair efficacy of rabbit knee joints. The scanning parameters were set as follows: spatial resolution of 9 µm pixel size; X-ray tube voltage at 65 kV; X-ray tube current of 380 mA; optical filter of Al 1 mm; scanning angular scope of 360°, and rotation step of 0.50°. The software GPUReconServer accompanied with the micro-CT system was utilized to reconstruct all resultant planograms under identical scanning parameters. The realistic three-dimensional (3D) visualization of knee joint specimens was performed by CTvox along with the micro-CT system. AFM Test of Surface Modulus and Surface Roughness of the Engineered or Regenerated Cartilage. Inspired by the nanomechanical test reported previously19, an atomic force microscope (AFM, Bruker Multimode8, USA) was employed to measure the surface modulus and the surface roughness of normal cartilage, neo-cartilage repaired under the 16-week implantation of cell-seeded and virgin bilayered PLGA/PLGA-HAp scaffold. Prior to the AFM test, the cartilage slices with 2 mm in length and 2 mm in width were gently peeled a thin layer from the experimental site, and the obtained cartilage slices were stuck on the specimen stage. Thereafter, the PeakForce Quantitative NanoMechanics (PeakForce QNM) mode with the tip type of RTESPA-300 (Bruker, USA) was carried out in the AFM test. Nanoscope Analysis from Bruker’s offline data processing software was used to calculate the surface modulus and the surface roughness of the engineered cartilage specimens. Tissue Retrieval and Histological Analysis. All experimental rabbits were sacrificed 12


Page 12 of 45

Page 13 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


by injecting the excess pentobarbital sodium at 8 and 16 weeks postoperatively. After harvesting and photographing the knee joint specimens, some specimens were fixed in 4% formalin and prepared for the micro-CT observation. All remaining samples were fixed in 4% formalin, decalcified in 30% formic acid, then embedded in paraffin, and sectioned at 4 µm by using a rotary microtome (Leica RM2235, Germany). Some sections were stained with haematoxylin and eosin (H&E), toluidine blue and safranin

O.

Moreover,

the

remaining

sections

were

further

collected

for

immunohistochemical assessment of collagen type II, which is the characteristic ECM component in hyaline cartilage. Western Blot to Detect the Relevant Protein Expression. Western Blot method was employed to determine the protein expression of p-smad 1, smad 2, collagen type I and collagen type II. According to manufacturer’s protocol, cells were rinsed by pre-cooling PBS three times, then cellular proteins were extracted with 400 µL mixture (1:100) of phenylmethanesulfonyl fluoride (PMSF) and RIPA lysis buffer (Medium, CoWin Biosciences Co., Ltd, China) under an ice bath. After 30 min, centrifugation was carried out at 12000 revolutions per minute at 4°C for 5 min, whereafter the supernatant was collected. Biquinolinic acid protein assay kit (BCA, CoWin Biosciences Co., Ltd, China) was employed to detect the concentration of the target protein. The pre-prepared protein and SDS-PAGE loading buffer (Reducing, 5×, CoWin Biosciences Co., Ltd, China) were mixed well in a proportion of 4:1, and then placed it into the boiling water bath for 5 min. Thereafter, an expeditious ice bathing was carried out until it was cooled down to room temperature. The sample was preserved at -20°C after a transitory centrifugation. According to the molecular weights of the target genes, a 10% gel was chosen to prepare the gelling system for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). PageRuler prestained protein ladder (#26616, 10 to 180 kDa, Thermo Scientific) and 40 µg pre-prepared protein sample conditioning fluid were added into the sample holders, and residual sample holders were added in the loading buffer (Reducing, 1×). Electrophoresis voltage was kept at 90 V. When the blue protein bands appeared, electrophoresis voltage was changed to 120 V. 13



Page 14 of 45

Thereafter, western transfer was carried out by a poly(vinylidene fluoride) (PVDF) membrane (Millipore, 0.45 µm) under the electric current of 200 mA for 70 min. 10% BSA was employed to seal the samples for 60 min. To detect the target proteins in the immunoprecipitate, SDS-PAGE and Western blot analyses were carried out with a 1:1000 dilution

of

primary

antibody

followed

by

a

1:5000

dilution

of

horseradish

peroxidase-conjugated anti-rabbit secondary antibody (Abcam). The expression level of each gene of interest was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Bioworld) with a 1:1000 dilution overnight at 4°C. Immunoprecipitated proteins were visualized by ECL reagent kit (Pointbio) with a 1:1 dilution in the chemiluminescence imager (3600, Clinx Science Instruments Co., Ltd. China). Western blot stripping buffer (CoWin Biosciences Co., Ltd, China) was used in a shaker for 30 min. The grayscales of Western blot bands were treated with a statistics software (Prism 5.0, GraphPad Software). Statistical Analysis. All quantitative results are expressed as mean ± standard deviation. Data were analyzed with one-way analysis of variance (ANOVA) or Student’s t-tests to examine the difference between the two samples. Statistically significant differences are performed by using “*” for p < 0.05, “**” for p < 0.01 and “***” for p < 0.001.

RESULTS Fabrication of Bilayered Composite Scaffolds. We used room-temperature compression molding and porogen leaching to fabricate PLGA porous scaffolds. Thereafter, the interior surface of the bony-layer PLGA scaffold was modified by HAp deposition. Ultimately, the required bilayered PLGA/PLGA-HAp composite scaffolds were fabricated by gluing the cartilage and subchondral layer scaffolds by CH2Cl2, as schematically presented in Figure 1c. Some as-fabricated cylindrical bilayered PLGA/PLGA-HAp scaffolds are shown in Figure 2a. The cylinder scaffolds were of diameter 4 mm and height of 5 mm, where the cartilage layer was of height of 1 mm, as demonstrated in Figure 2b. The cementing line of the bilayered PLGA/PLGA-HAp scaffolds is also indicated in Figure 2b. The upper and lower layers were connected very tightly in our bilayered porous scaffolds. 14


Page 15 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2. Global observations (a and b) and SEM microscopic morphology (c) of as-prepared bilayered scaffolds. The dashed yellow line in (c) indicates the border of the cartilage and subchondral bone layers.

SEM was employed to observe the interior pore structure and morphology of the bilayered PLGA/PLGA-HAp scaffolds. The yellow dashed line in Figure 2c represents the cementing line or boundary between the upper cartilage layer and under the subchondral bone layer. Because slight extrusion was performed when gluing the scaffolds, a few pores near the dashed line became squashed (Figure S1), even got compaction. The remaining pores had a rational pore sizes as we controlled with porogen. A magnified view (Figure S2) illustrates relatively smooth pore walls of the cartilage-layer scaffold and a lot of HAp deposition covering on the interior surface of the bony-layer scaffold. The porosity and pore size were well controlled by the fraction and size of sodium chloride porogen. The resultant cartilage-layer scaffolds were of pore sizes 100 - 200 µm, while those of the subchondral bone layer were of pore sizes 300 - 450 µm. We determined the porosities of cartilage-layer and subchondral-layer scaffolds by a modified liquid replacement method, as calculated by Eq. (1). The results are shown in Figure 3. The porosity of the cartilage layer scaffold was about 92%; the subchondral layer PLGA scaffold had porosity about 77%. The porosity of the PLGA scaffold with HAp modification was about 76%, which was calculated with Eq. (2).

15



Figure 3. Porosities of cartilage layer and subchondral layer scaffolds. n = 5 for each group.

The deposition on the interior surface of the subchondral bone layer scaffold was further characterized. FESEM-EDS were employed to qualitatively detect the calcium-phosphorus ratio of deposition. As demonstrated in Figure 4a, elements calcium and phosphorus were detected on the bony-layer PLGA/HAp scaffold. The peak area of each element was calculated, and the calcium-phosphorus ratio was obtained as 1.69, which was close the theoretical calcium-phosphorus ratio of hydroxyapatite 1.67.69-70 Figure 4a shows also the images of the elements carbon, oxygen, calcium and phosphorus, confirming the success of the deposition on the interior surfaces of scaffolds. Natural HAp is an inorganic crystal.71 Thereby, XRD was employed to detect the diffraction peaks of the deposition on the interior surface of the subchondral-layer PLGA scaffold. We used a commercial HAp (Sinopharm Chemical Reagent Co., Ltd, China) as standard, and proved that the deposition is a kind of analogous hydroxyapatite (Figure 4b). Thereinto, the characteristic diffraction peaks of HAp were observed at the 2θ angle of 27.4° and 31.8°. They were not as sharp as the characterized diffraction peaks of commercial hydroxyapatite, which illustrated that the deposited HAp was of just a moderate degree of crystallization. Thereafter, TGA technology was employed, taking advantage of difference of heat stabilities of polymer and ceramics. The fraction of HAp deposition on the interior surfaces of the subchondral-layer PLGA scaffold was determined to be 13.9%, as shown in Figure 4c. 16


Page 16 of 45

Page 17 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4. Qualitative and quantitative characterization of hydroxyapatite deposition on the interior surface of the subchondral-layer PLGA scaffold. (a) FESEM-EDS analysis; (b) XRD patterns of the HAp deposition on the interior surface of subchondral-layer PLGA scaffold and commercial HAp; (c) quantitative test of HAp deposition on the interior surface of subchondral-layer PLGA scaffold by TGA.

Mechanical Properties of Bilayered Scaffolds. The mechanical properties of the scaffolds at “dry” and “wet” state were also characterized. Figure 5a schematically shows the process of compression testing for the bilayered scaffold. Compressive modulus (E) is generally used to characterize the mechanics of a porous scaffold. Thereinto, E was determined as the slope of the initial linear section of the stress-strain curve. The stress-strain curve of the bilayered scaffold exhibited two scopes in Figure 5b, resulting in E1 (2.8 ± 1.7 MPa) and E2 (20.3 ± 0.7 MPa) representing the compression moduli 17



of the “wet-state” cartilage layer and subchondral bone layer, respectively. The porosity plays a key role on the compression modulus of a porous scaffold. For the remaining scaffolds, only one elastic deformation region was demonstrated in their stress-strain curves due to their homogeneous porosity. According to Figure 5c, the compression moduli of the “wet-state” cartilage-layer scaffold (PLGA) were 0.4 ± 0.1 MPa; the compression moduli of the “wet-state” subchondral-layer scaffold (PLGA) were 5.6 ± 0.3 MPa; the compression moduli of the “wet-state” subchondral-layer scaffold (PLGA/HAp) were 12.4 ± 1.0 MPa. It is not difficult to understand that the HAp deposition enhanced the mechanical property of the bony-layer scaffold. The mechanical properties of the scaffolds at “dry” state are showed in Figure S3a and S3b. We also made normalization of the data at the “wet-state” over those at the “dry-state” with respect to each group, and then integrated the normalized vales from all of the groups into Figure S3c. Despite of hydrophobicity of PLGA, the “wet-state” moduli were significantly less than the “dry-state” data, consistent with our previous finding62. The present study indicated that about 80% moduli remained after the wetting process. Since tissue engineering or tissue induction scaffolds are eventually used in a wet aqueous environment, such a “discount” should be taken into consideration in a biomaterial design based on merely “dry-state” mechanical data. Nevertheless, Figure 5 in the main manuscript and Figure S3 in Supporting Information exhibited similar trends of the modulus change among different groups, and thus “dry” or “wet” did not alter the basic conclusions of comparison between different scaffold groups with PLGA as the basic matrix.

18


Page 18 of 45

Page 19 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 5. Mechanical properties of the as-fabricated PLGA porous scaffolds at “wet” state. (a) Schematic presentation of the compressing process of the bilayered PLGA/PLGA-HAp scaffold. E1 represents the compressive modulus of the cartilage layer and E2 for the subchondral layer; (b) typical stress-strain curves of four groups of scaffolds with the “wet” state; (c) The compressive moduli of the indicated four groups of scaffolds with the “wet” state. n = 5 for each group. Corresponding “dry-state” data are shown in Figure S3. 19



In Vitro Cell Adhesion on the Interior Surfaces of the Porous Scaffolds. Besides physicochemical characterization of the scaffolds, we seeded the vigorous MSCs onto scaffolds for the cartilage layer (PLGA) and subchondral bone layer (PLGA or PLGA/HAp) to assess cell adhesion and proliferation in vitro. After 7-day culture in the basal medium, MSCs were fixed on the interior surface of the scaffold samples, and then F-actins and nuclei of MSCs were fluorescently stained. Thereafter, an inverted fluorescence microscope was employed to observe the immunofluorescence staining MSCs, with some images demonstrated in Figure 6.

Figure 6. Fluorescence micrographs of MSCs after 7 days of culture in the basal medium on cartilage layer and subchondral layer scaffolds.

Cells adhered on the interior surfaces of the porous scaffolds very well in all of groups. Besides these fluorescence micrographs in the main manuscript, a typical SEM image is shown in Figure S4, which demonstrated further a satisfactory MSC growth on the interior surface of the scaffolds. Hence, we confirmed partially in vitro biocompatibility of both 20


Page 20 of 45

Page 21 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


layers of our composite scaffolds. In Vivo Osteochondral Repair with a Rabbit Model. In the in vivo experiments, a defect with 4 mm in diameter and 5 mm in depth was created in the media femoral condyle by applying a surgical drill bit, as demonstrated in Figure 7a. After one-week MSC culture on the cartilage layer of bilayered PLGA/PLGA-HAp scaffold, the cell-seeded and virgin bilayered PLGA/PLGA-HAp scaffolds were implanted into the rabbit knee joints to examine the efficacies of tissue engineering and tissue regeneration, respectively. After 16 weeks, a macroscopic specimen examination of knee joints was carried out to make sure of no inflammation of the synovial membrane and other joint tissues. In the blank control group (Figure 7b), compared to the normal osteochondral site on the left, the defect on the right was covered with a few amorphous soft fibrous tissue around the interior walls, yet an obvious vacancy could still be seen in the lateral condyle at 16-week postoperation. In the group of virgin bilayered PLGA/PLGA-HAp scaffolds (Figure 7c and 7e), the vast majority defects were filled with regenerated tissue after 16-week implantation, and some bone trabecula were found in the defect site via the micro-CT observation. In the cell-seeded bilayered PLGA/PLGA-HAp scaffold group (Figure 7d and 7f), all defects were repaired completely with a cartilage-like tissue, which was smooth as the normal cartilage, and the demarcation integrated with the surrounding tissue was not visible. The bone trabeculas were uniformly distributed in the defect via the micro-CT observation, indicating repair of cartilage and subchondral bone simultaneously.

21



Figure 7. Observation of the osteochondral defects and repair effects in rabbit knee joints. (a) A defect was created in the media femoral condyle by applying a surgical drill bit; the defect was 4 mm in diameter and 5 mm in thickness in the medial condyles of the knee joint and the medial condyle defect was implanted with scaffold; (b) the knee joint defects without implanting any scaffold after 16 weeks; (c) the knee joint implanted with PLGA/PLGA-HAp bilayered scaffolds after 16 weeks, and (e) its micro-CT observation; (d) the knee joint implanted with PLGA/PLGA-HAp bilayered scaffolds with MSCs after 16 weeks, and (f) corresponding micro-CT images.

Biomechanical Tests as well as Surface Roughness Observations of Regenerated Cartilage with AFM. The biomechanical property of the 16-week regenerated cartilage is a significant proof for assessing the repair ability. Considering that it is not easy to dig out the regenerated tissue and make its shape regular from the defect site, we employed AFM to measure the surface modulus and the surface roughness of the regenerated cartilage on the appropriate length scales. 22


Page 22 of 45

Page 23 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figures 8a and 8b show the distribution of surface modulus and roughness of the normal and regenerated cartilages. The Young's moduli of 16-week regenerated cartilage of the cell-seeded bilayered PLGA/PLGA-HAp scaffold, the virgin bilayered PLGA/PLGA-HAp scaffold and normal cartilage were, respectively, in a range of 6 - 10 GPa, 3 - 9 GPa and 7 11 GPa, which consists with the data for joint cartilage19. In Figure 8b, Nanoscope Analysis resulted in the surface roughness of 16-week regenerated cartilage of the cell-seeded bilayered PLGA/PLGA-HAp scaffold, virgin bilayered PLGA/PLGA-HAp scaffold and normal cartilage. They read 10.4 ± 2.6 nm, 19.6 ± 5.3 nm and 8.4 ± 0.4 nm, respectively. Taking all the above data into consideration, it is conclusive that the virgin bilayered PLGA/PLGA-HAp scaffold repaired most of the cartilage defect, and the seeding of external cells made some additional improvement.

Figure 8. AFM test of surface modulus (a) and surface roughness (b) of normal cartilage, 23



neo-cartilages repaired under the implantation of PLGA/PLGA-HAp bilayered scaffold and PLGA/PLGA-HAp bilayered scaffold with MSCs after 16 weeks. n = 3 for each group.

Histological Examinations of the Regenerated Tissues to Show Characteristics of Cartilage and Subchondral Bone. To assess the tissue response to the implanted bilayered scaffolds and the defect-repairing progress, histological examinations were carried out. No significant inflammatory response was observed in the histological examination among all of groups. In the blank control group, the staining results showed just a small amount of irregular fibrous tissues in the subchondral region, and the obvious vacancy could still be seen at 16-week postoperation, as demonstrated in Figure 9a. In the PLGA/PLGA-HAp group, most of the defects were padded with regenerated tissue mixed with immature cartilage in the chondral layer, and new bone was formed in the subchondral region. Hence, our bilayered PLGA/PLGA-HAp scaffold triggered a significant healing progress although a handful of residual scaffold was still perceived in the histological examination after a 16-week implantation. In the group of PLGA/PLGA-HAp with MSCs, the defects were filled almost completely after 16 weeks. A higher percentage of hyaline cartilage in the chondral layer and the bone regeneration in the subchondral region, which were well integrated with native tissue in the site. The high-magnification observations after H&E, safranin O and toluidine blue staining demonstrated that ECM was actively secreted in the virgin and cell-seeded groups. Oval-shaped cartilage lacuna was clearly formed, which is a very important characteristic of hyaline cartilage (Figure 9b). Another critical characteristic of cartilage is the expression of type II collagen. Immunohistochemical tests were thus employed to detect the expression of such a type of collagen in the specimen sections of the virgin and cell-seeded groups, as shown in Figure S5. While a slightly lower expression level of type II collagen was found for the virgin group, both groups exhibited similar expression levels of type II collagen to the normal group.

24


Page 24 of 45

Page 25 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 9. Histological images of reparative tissues after 16-week implantation of PLGA/PLGA-HAp and PLGA/PLGA-HAp with MSCs. (a) H&E staining (HE), safranin O staining (SO) and toluidine blue staining (TB). The yellow arrows in (a) indicate the initial defect sites. (b) high-magnification images to better visualize cartilage lacunae as a 25



characteristic of hyaline cartilage.

Western Blot Analysis of Expression of Characteristic Proteins in the Regenerated Tissues Extracted from the Implanting Sites of the Bilayered Composite Scaffolds. Western blot was used to semi-quantify the target protein expression in neo-tissues repaired by the virgin and cell-seeded bilayered PLGA/PLGA-HAp scaffolds after 16-week implantation. Thereinto, protein expressions of p-smad 1, smad 2, collagens (type I and type II) were determined (Figure 10). No matter in the virgin group or cell-seeded group, the relevant proteins remained on an upward expression from 8 weeks to 16 weeks. As for the relative level of proteins p-smad 1, smad 2, type I collagen and type II collagen, we observed a higher expression in the cell-seeded PLGA/PLGA-HAp group than in the virgin group. There is an inversion of the expression levels of type II collagen between the virgin and cell-seeded groups at 16 weeks.

Figure 10. Western blot analysis. (a) Protein bands presentation of p-smad 1, smad 2, collagen type I and collagen type II; (b) Analysis of gray scale values of those protein expression in Western blot. The asterisk indicates significant difference between the examined two groups. n = 4 for each group.

26


Page 26 of 45

Page 27 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


DISCUSSION Tissue Regeneration or Tissue Induction Affords, like Tissue Engineering, an Important Strategy to Regenerate Cartilage and Subchondral Bone Based on Appropriate Scaffolds. While the concept of tissue engineering using porous scaffolds and external seed cells to engineer a tissue is a milestone in medicine72-74, tissue regeneration or tissue induction is another strategy using only implanted biodegradable and bioactive materials and recruiting internal cells75-84. Both strategies are very helpful for regenerative medicine. While much progress has been made in the tissue regenerative biomaterials85-91, the present study develops a facile biomaterial technique to prepare a bilayered composite scaffolds composed of two FDA-approved biomaterials, a biodegradable polymer PLGA and a bioactive ceramic HAp. The repair of articular cartilage defects remains a rock-ribbed challenge considering that natural articular cartilage lacks self-healing capacity and ideal clinical treatments. Tissue engineering and regenerative medicine emerged as an alternative strategy with great promise for articular cartilage regeneration.92 Driven by increasingly clinical needs, there have been many studies on articular cartilage repair. In recent years, bilayered porous scaffolds have been raised to simultaneously mimic interior structures and physiological properties of cartilage and subchondral bone.93-94 Cell-seeded bilayered scaffolds probably have a better repairing capacity than the cell-free ones. However, the introduction of extra cells led to much restriction such as long-time preparation, complicated examination and expensive regulatory approval of applications because of the cost and the immunogenic problems. The regulatory disadvantages thereby enhance interest in virgin bilayered scaffolds for articular cartilage repair. The mechanical properties and interior structural optimization are still a big challenge for those virgin bilayered scaffolds. So we are highly interested in appropriate bilayered scaffolds for osteochondral tissue engineering and tissue regeneration. The Bilayered PLGA/PLGA-HAp Composite Scaffold in This Study Affords One of Appropriate Biomaterial Systems. In this study, a bilayered PLGA/PLGA-HAp composite scaffold was fabricated by combining room-temperature compression molding and particle leaching, plasma-treated surface deposition, and solvent sticking methods to simultaneously regenerate cartilage and subchondral bone. PLGA and HAp are both approved by FDA for 27



clinical applications.28,

95-96

In addition to the bilayered design and elaborate material

selection, porosity and pore size of a bilayered scaffold were well controlled by the fraction and size of porogen. As reported previously for tissue engineering26-27, the cartilage layer scaffold with a porosity of 92% and a pore size of 100 - 200 µm are thought to promote chondrogenesis, and the subchondral bone layer scaffold with a porosity of 77% and a pore size of 300 - 450 µm are recommended for promoting vascularization and bone formation. These pore sizes and porosity were taken in the present study. We succeeded in fabrication of the expected bilayered PLGA/PLGA-HAp composite scaffolds, as demonstrated in Figures 2 and 3. The mould was designed at a diameter of 4 mm, and there was an unconspicuous scaffold contraction because of solvent evaporation. If we have a close-up view of the cementing line between the cartilage layer and the subchondral layer, it is not difficult to observe that a few pores became squashed, even got compaction at a thickness of about 85 µm (Figure S1), due to compression when upper and down layer scaffolds were glued. These slight squash and compaction made the two layers connected tightly, and became a positive influence factor of mechanical properties for the bilayered scaffolds. As shown in Figure 5, the combined efficiency of the mechanical properties of bilayered scaffolds was greater than the sum of each single-layer scaffold used in isolation. MSCs have recently aroused the concern of fundamental research.97-98 For the in vitro study of biocompatibility, F-actins and nuclei of 7-day cultural MSCs in the bilayered PLGA/PLGA-HAp scaffolds were fluorescently stained, and we did observe a significant cell growth in the porous scaffolds. The New Zealand white rabbit model was chosen for our in vivo experiments. MSCs were employed to inject onto the cartilage layer. As for the cell-seeded bilayered PLGA/PLGA-HAp scaffold group, the 16-week follow-up results indicated a good spontaneous healing of the osteochondral defect on the biomechanics and morphological character, as shown in Figures 7, 8 and 9. After a 16-week implantation of the virgin bilayered PLGA/PLGA-HAp scaffold into the osteochondral defect, most of repair was achieved. And the scaffold seeded with MSCs led to an additional repair. Both the groups with porous scaffolds behaved much better than the untreated group. No matter for the proportion and biomechanics of regenerated tissue, or 28


Page 28 of 45

Page 29 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


the morphology of neo-chondrocytes (Figures 7, 8 and 9), osteochondral defects treated with a virgin bilayered PLGA/PLGA-HAp scaffold alone or a bilayered PLGA/PLGA-HAp scaffold associated with MSCs showed a similar cartilaginous and subchondral formation. So it is the bilayered PLGA/PLGA-HAp scaffold that mainly contributed to the progress of cartilage and subchondral bone regeneration. This finding is consistent with some previous studies.99-100 In addition, Western blot was employed to semi-quantify the target protein expression of p-smad 1, smad 2, collagen type I and collagen type II in neo-tissue repaired by the cell-seeded and virgin bilayered PLGA/PLGA-HAp scaffolds after a 16-week implantation (Figure 10a). It has been known that the levels of gene expressions of p-smad 1 and type I collagen were positively related to osteogenesis;101-103 it has also been reported that the levels of gene expressions of smad 2 and type II collagen were positively related to chondrogenesis.104-105 As showed in Figure 10b, the levels of the relevant protein expression in the virgin group and cell-seeded group remained on a consistent and upward trend. Although most protein expression had a higher level for the cell-seeded group, there was a slight inversion on the level of protein expression of type II collagen in 16-week implantation of virgin bilayered PLGA/PLGA-HAp scaffolds, indicating the potential of the composite bilayered scaffold in osteochondral regeneration. The Composite Scaffold is Appropriate Owing to Its Biomechanical and Chemical Microenvironment Mimics to Cartilage and Subchondral Bone. There are different moduli, physical structures and physiological functions between articular cartilage and subchondral bone, therefore, we mimicked their biomechanical requirements to fabricate a bilayered composite scaffold with different porosities and pore sizes. Moreover, the mechanical properties under the “wet” state as simulated physiological environment should be taken into consideration, especially for the biomaterial design of in-situ tissue induction. In this study, the mechanical properties of cartilage-layer PLGA scaffold and subchondral-layer PLGA/HAp scaffold of our bilayered scaffold were tested at their “dry” and “wet” states, and the results showed a befitting biomechanic matching for the as-fabricated bilayered composite scaffold. According to the studies reported previously, the instantaneous compressive Young’s 29



Page 30 of 45

modulus of articular cartilage is 1.36 - 39.2 MPa, and the compressive modulus of subchondral bone is among 1.4 - 9800 MPa.27, 106-107 The cartilage layer made of PLGA and the subchondral bone layer made of PLGA/HAp in the bilayered scaffold at the “wet-state” were of moduli 2.8 ± 1.7 MPa and 20.3 ± 0.7 MPa, respectively. Our bilayered PLGA/PLGA-HAp composite scaffolds exhibited biomechanic matching. Moreover, we also tested the biomechanical properties of 16-week regenerative articular cartilage, showing an applicable result with a comparison of normal articular cartilage. Obviously, a favourable biomechanical environment afforded not only a supportive interconnective structure but also a biomimetic mechanical stimulation, which is greatly beneficial to the growth of cells and tissues. It has been reported that surface chemistry and physics of scaffolds can prominently regulate cell behaviors.108-111 One of our previous work68 illustrated that chemical functional groups on the surface of biomaterials can regulate stem cell differentiation via tuning protein adsorption, then nonspecific cell adhesion and thus cell spreading. We think that the HAp deposition on the interior surface of scaffold for the subchondral bone might be helpful for cell adhesion, differentiation and bone matrix formation.48-49,

77, 79, 112

MSCs could be

triggered to facilitate the formation of tissue cells for matching the microenvironments.24-25 MSCs seeded into the cartilage layer and subchondral layer can facilitate the formation of cells towards cartilage and subchondral bone, respectively. Our HAp coating can afford a better surficial chemistry micro-circumstance to drive the seed cells into the subchondral bone differentiation. It is thus reasonable that the cartilage and subchondral bone were restored well under the implantation the cell-seeded bilayered PLGA/PLGA-HAp scaffold. A converse speculation is not hard to be made. If the tissue induction method is employed for osteochondral repair, even though exogenous seed cells are not used, endogenous chondrocyte, osteocyte and MSCs are presumably guided to migrate into the defect sites, and MSCs might be triggered to facilitate the formation of chondrocytes and subchondral bone cells. New tissues could be formed along with the secretion of the corresponding ECM and the appearance of the corresponding histological morphology. Taking all the results using the virgin scaffolds into consideration, it is likely that this tissue regeneration process was mediated by MSCs recruited from the subchondral bone penetrating 30


Page 31 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


into the scaffolds towards autologous matrix-induced chondrogenesis, similar to a previously proposed “cell homing” concept.113-117 Limitations of the Present Studies. Several limitations in this study have to be mentioned. Only a 16-week follow-up period was examined after the implantation in vivo, so that the results obtained by using merely the porous scaffold cannot have an identical repair effect to the cell-seeded group. Another critical argument comes from the probable resorption of neo-cartilage or neo-bone formed via tissue engineering or tissue regeneration after 3 - 5 years.118-120 So a long-term follow-up study of outcomes is needed for the newly regenerated tissue, in particular, to examine the phenotype of chondrocytes and the biomechanical properties of the tissue. The larger animal model, which closely mimics the loading conditions of human knee joints, are also necessary to be considered.121-122 Anyway it is attractive if the bilayered scaffold has the ability to induce osteochondral tissue reestablishment without necessarily seeding cells. Firstly, the biological and ethical problems related to the cells culture would be no longer in existence. Meanwhile, free of cells helps a porous scaffold tremendously cut its costs.123 More importantly, it could be fabricated as an off-the-shelf and easy-to-use product for clinical applications.

CONCLUSIONS Bilayered PLGA/PLGA-HAp composite scaffolds were fabricated after combination of room-temperature compression-molding/porogen-leaching to prepare PLGA scaffolds for each layer of the cartilage and the subchondral bone, the bioceramic deposition of the interior pore layer to obtain the PLGA-HAp scaffold for the subchondral bone layer, and finally to use an organic solvent to stick the two layers. The bilayered PLGA/PLGA-HAp composite scaffold was found to repair an osteochondral defect with the simultaneous formation of cartilage and subchondral bone both with and without MSCs as seed cells. While the introduction of the extra cells led to better results, the bilayered composite scaffold exhibited satisfactory in vivo efficacy after implanted into the rabbit knee joints for 16 weeks. According to the biomechanical analysis, micro-CT observations, histological observations and relative expression levels of characteristic proteins via Western blot, the bilayered PLGA/PLGA-HAp composite scaffold exhibited a favorable regeneration of osteochondral 31



defect. The biomechanics of neo-tissues characterized by AFM basically matched with that of native tissue. This study illustrates that the bilayered PLGA/PLGA-HAp scaffold is appropriate for osteochondral tissue engineering and tissue regeneration, and even a tissue induction after implanting the composite scaffolds without seeding cells is available for in vivo osteochondral regeneration.

SUPPLEMENTARY INFORMATION The Supporting Information is available free of charge on the ACS Publications website at DOI: ***. SEM images to present the high-magnification border of the cartilage and subchondral layers; SEM observations of the subchondral bone layer before and after HAp coating on the interior surfaces of PLGA scaffolds; SEM images of MSCs on the scaffolds after 7-day cell culture; Mechanical properties of the as-fabricated PLGA porous scaffolds at “dry” state and the normalized moduli of the as-fabricated four groups of scaffolds at both “dry” and “wet” states; Histological images of reparative tissues after 16-week implantation of bilayered PLGA/PLGA-HAp scaffolds alone and seeded with MSCs (staining of type II collagen).

CORRESPONDING AUTHOR *

E-mail: [email protected].

NOTES The authors declare no competing financial interest.

ACKNOWLEDGMENTS The authors are grateful for the financial supports from National Key R&D Program of China (grant No. 2016YFC1100300), National Science Foundation of China (grants No. 81401790, 51533002, 51603044), Science and Technology Commission of Shanghai Municipality (grant

32


Page 32 of 45

Page 33 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


No. 17JC1400200), and Natural Science Foundation of Jiangxi Province (grant No. 20171ACB21057, 20151BAB205053).

33



REFERENCES (1) Swieszkowski, W.; Tuan, B. H.; Kurzydlowski, K. J.; Hutmacher, D. W., Repair and Regeneration of Osteochondral Defects in the Articular Joints. Biomol. Eng. 2007, 24 (5), 489-495. DOI: 10.1016/j.bioeng.2007.07.014. (2) Temenoff, J. S.; Mikos, A. G., Review: Tissue Engineering for Regeneration of Articular Cartilage. Biomaterials 2000, 21 (5), 431-440. DOI: 10.1016/S0142-9612(99)00213-6. (3) Huey, D. J., Unlike Bone, Cartilage Regeneration Remains Elusive. Science 2012, 338 (6109), 917-921. DOI: 10.1126/science.1222454. (4) Makris, E. A.; Gomoll, A. H.; Malizos, K. N.; Hu, J. C.; Athanasiou, K. A., Repair and Tissue Engineering Techniques for Articular Cartilage. Nat. Rev. Rheumatol. 2015, 11 (1), 21-34. DOI: 10.1038/nrrheum.2014.157. (5) Kalson, N. S.; Gikas, P. D.; Briggs, T. W., Current Strategies for Knee Cartilage Repair. Int. J. Clin. Pract. 2010, 64 (10), 1444-1452. DOI: 10.1111/j.1742-1241.2010.02420.x. (6) Ringe, J.; Burmester, G. R.; Sittinger, M., Regenerative Medicine in Rheumatic Disease Progress in Tissue Engineering. Nat. Rev. Rheumatol. 2012, 8 (8), 493-8. DOI: 10.1038/nrrheum.2012.98. (7) Foss, C.; Merzari, E.; Migliaresi, C.; Motta, A., Silk Fibroin/Hyaluronic Acid 3D Matrices for Cartilage Tissue Engineering. Biomacromolecules 2013, 14 (1), 38-47. DOI: 10.1021/bm301174x. (8) Eslahi, N.; Abdorahim, M.; Simchi, A., Smart Polymeric Hydrogels for Cartilage Tissue Engineering: A Review on the Chemistry and Biological Functions. Biomacromolecules 2016, 17 (11), 3441-3463. DOI: 10.1021/acs.biomac.6b01235. (9) Radhakrishnan, J.; Subramanian, A.; Krishnan, U. M.; Sethuraman, S., Injectable and 3D Bioprinted Polysaccharide Hydrogels: From Cartilage to Osteochondral Tissue Engineering. Biomacromolecules 2016, 18 (1), 1-26. DOI: 10.1021/acs.biomac.6b01619. (10) Minas, T.; Gomoll, A. H.; Rosenberger, R.; Royce, R. O.; Bryant, T., Increased Failure Rate of Autologous Chondrocyte Implantation After Previous Treatment With Marrow Stimulation Techniques. Am. J. Sports Med. 2009, 37 (5), 902-908. DOI: 10.1177/0363546508330137. (11) Martin, I.; Miot, S.; Barbero, A.; Jakob, M.; Wendt, D., Osteochondral Tissue Engineering. J. Biomech. 2007, 40 (4), 750-765. DOI: 10.1177/0363546508330137. (12) Newman, A. P., Articular Cartilage Repair. Am. J. Sports Med. 1998, 26 (2), 309-324. DOI: 10.1177/03635465980260022701. 34


Page 34 of 45

Page 35 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(13) O'Shea, T. M.; Miao, X., Bilayered Scaffolds for Osteochondral Tissue Engineering. Tissue Eng., Part B 2008, 14 (4), 447-464. DOI: 10.1089=ten.teb.2008.0327. (14) Chen, G. P.; Sato, T.; Tanaka, J.; Tateishi, T., Preparation of a Biphasic Scaffold for Osteochondral Tissue Engineering. Mater. Sci. Eng., C 2006, 26 (1), 118-123. DOI: 10.1016/j.msec.2005.07.024. (15) Shao, X. X.; Goh, J. C.; Hutmacher, D. W.; Lee, E. H.; Ge, Z. G., Repair of Large Articular Osteochondral Defects Using Hybrid Scaffolds and Bone Marrow-Derived Mesenchymal Stem Cells in a Rabbit Model. Tissue Eng., Part A 2006, 12 (6), 1539-1551. DOI: 10.1089/ten.2006.12.1539. (16) Mano, J. F.; Reis, R. L., Osteochondral Defects: Present Situation and Tissue Engineering Approaches. J. Tissue Eng. Regener. Med. 2007, 1 (4), 261-273. DOI: 10.1002/term.37. (17) Giannoni, P.; Lazzarini, E.; Ceseracciu, L.; Barone, A. C.; Quarto, R.; Scaglione, S., Design and Characterization of a Tissue-Engineered Bilayer Scaffold for Osteochondral Tissue Repair. J. Tissue Eng. Regen. Med. 2015, 9 (10), 1182-1192. DOI: 10.1002/term.1651. (18) Lee, Y. H.; Wu, H. C.; Yeh, C. W.; Kuan, C. H.; Liao, H. T.; Hsu, H. C.; Tsai, J. C.; Sun, J. S.; Wang, T. W., Enzyme-Crosslinked Gene-Activated Matrix for the Induction of Mesenchymal Stem Cells in Osteochondral Tissue Regeneration. Acta Biomater. 2017, 63, 210-226. DOI: 10.1016/j.actbio.2017.09.008. (19) Zhang, K. X.; Yan, S. F.; Li, G. F.; Cui, L.; Yin, J. B., In-situ Birth of MSCs Multicellular Spheroids in Poly(L-glutamic acid)/Chitosan Scaffold for Hyaline-like Cartilage Regeneration. Biomaterials 2015, 71, 24-34. DOI: 10.1016/j.biomaterials.2015.08.037. (20) Carmont, M. R.; Careysmith, R.; Saithna, A.; Dhillon, M.; Thompson, P.; Spalding, T., Delayed Incorporation of A TruFit Plug: Perseverance is Recommended. Arthroscopy 2009, 25 (7), 810-814. DOI: 10.1016/j.arthro.2009.01.023. (21) Bekkers, J. E. J.; Bartels, L. W.; Vincken, K. L.; Dhert, W. J. A.; Creemers, L. B.; Saris, D. B. F., Articular Cartilage Evaluation After TruFit Plug Implantation Analyzed by Delayed Gadolinium-Enhanced MRI of Cartilage (dGEMRIC). Am. J. Sports Med. 2013, 41 (6), 1290-1295. DOI: 10.1177/0363546513483536. (22) Quarch, V. M. A.; Enderle, E.; Lotz, J.; Frosch, K. H., Fate of Large Donor Site Defects in Osteochondral Transfer Procedures in the Knee Joint with and without TruFit Plugs. Arch. Orthop. Trauma Surg. 2014, 134 (5), 657-666. DOI: 10.1007/s00402-014-1930-y. (23) Kon, E.; Filardo, G.; Robinson, D.; Eisman, J. A.; Levy, A.; Zaslav, K.; Shani, J.; Altschuler, N., Osteochondral Regeneration Using a Novel Aragonite-Hyaluronate Bi-phasic Scaffold in A Goat Model. Knee Surg. Sport Tr. A. 2014, 22 (6), 1452-1464. DOI: 10.1007/s00167-013-2467-2. 35



(24) Chen, J. W.; Wang, C. Y.; Lü, S. H.; Wu, J. Z.; Guo, X. M.; Duan, C. M.; Dong, L. Z.; Song, Y.; Zhang, J. C.; Ding, J. D.; Li, D. X., In Vivo Chondrogenesis of Adult Bone-Marrow-Derived Autologous Mesenchymal Stem Cells. Cell Tissue Res. 2005, 319 (3), 429-438. DOI: 10.1007/s00441-004-1025-0. (25) Uematsu, K.; Hattori, K.; Ishimoto, Y.; Yamauchi, J.; Habata, T.; Takakura, Y.; Ohgushi, H.; Fukuchi, T.; Sato, M., Cartilage Regeneration Using Mesenchymal Stem Cells and A Three-Dimensional Poly-lactic-glycolic acid (PLGA) Scaffold. Biomaterials 2005, 26 (20), 4273-4279. DOI: 10.1016/j.biomaterials.2004.10.037. (26) Duan, P. G.; Pan, Z.; Cao, L.; He, Y.; Wang, H. R.; Qu, Z. H.; Dong, J.; Ding, J. D., The Effects of Pore Size in Bilayered Poly(lactide-co-glycolide) Scaffolds on Restoring Osteochondral Defects in Rabbits. J. Biomed. Mater. Res., Part A 2014, 102 (1), 180-192. DOI: 10.1002/jbm.a.34683. (27) Pan, Z.; Duan, P. G.; Liu, X. N.; Wang, H. R.; Cao, L.; He, Y.; Dong, J.; Ding, J. D., Effect of Porosities of Bilayered Porous Scaffolds on Spontaneous Osteochondral Repair in Cartilage Tissue Engineering. Regener. Biomater. 2015, 2 (1), 9-19. DOI: 10.1093/rb/rbv001. (28) Wang, D. X.; He, Y.; Bi, L.; Qu, Z. H.; Zou, J. W.; Pan, Z.; Fan, J. J.; Chen, L.; Dong, X.; Liu, X. N.; Pei, G. X.; Ding, J. D., Enhancing the Bioactivity of Poly(lactic-co-glycolic acid) Scaffold with A Nano-Hydroxyapatite Coating for the Treatment of Segmental Bone Defect in A Rabbit Model. Int. J. Nanomed. 2013, 8, 1855-1865. DOI: 10.2147/IJN.S43706. (29) Wang, Z.; Zhang, Z.; Zhang, J. C.; She, Z. J.; Dong, D. J., Distribution of Bone Marrow Stem Cells in Large Porous Polyester Scaffolds. Chin. Sci. Bull. 2009, 54 (17), 2968-2975. DOI: 10.1007/s11434-009-0181-8. (30) Yu, L.; Feng, Y. K.; Li, Q.; Hao, X. F.; Liu, W.; Zhou, W.; Shi, C. C.; Ren, X. K.; Zhang, W. C., PLGA/SF Blend Scaffolds Modified with Plasmid Complexes for Enhancing Proliferation of Endothelial Cells. React. Funct. Polym. 2015, 91-92, 19-27. DOI: 10.1016/j.reactfunctpolym.2015.04.003. (31) Qi, R. L.; Tian, X. J.; Guo, R.; Luo, Y.; Shen, M. W.; Yu, J. Y.; Shi, X. Y., Controlled Release of Doxorubicin from Electrospun MWCNTs/PLGA Hybrid Nanofibers. Chin. J. Polym. Sci. 2016, 34 (9), 1047-1059. DOI: 10.1016/j.reactfunctpolym.2015.04.003. (32) Duo, X. H.; Li, Q.; Wang, J.; Lv, J.; Hao, X. F.; Feng, Y. K.; Ren, X. K.; Shi, C. C.; Zhang, W. C., Core/Shell Gene Carriers with Different Lengths of PLGA Chains to Transfect Endothelial Cells. Langmuir 2017, 33 (46), 13315-13325. DOI: 10.1021/acs.langmuir.7b02934. (33) Deng, Y.; Zhang, M. J.; Chen, X. C.; Pu, X. M.; Liao, X. M.; Huang, Z. B.; Yin, G. F., A Novel Akermanite/poly (lactic-co-glycolic acid) Porous Composite Scaffold Fabricated Via a Solvent Casting-Particulate Leaching Method Improved by Solvent Self-Proliferating Process. Reg. Biomater. 2017, 4 (4), 233-242. DOI: 10.1093/rb/rbx014. 36


Page 36 of 45

Page 37 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(34) Huang, Y.; Wang, S. T.; Zhu, X. Y., Recent Advances of Biodegradable Hyperbranched Polymers. Acta Polym. Sin. 2017, (2), 245-258. DOI: 10.1093/rb/rbx014. (35) Peng, Y. M.; Liu, Q. J.; He, T. L.; Ye, K.; Yao, X.; Ding, J. D., Degradation Rate Affords a Dynamic Cue to Regulate Stem Cells Beyond Varied Matrix Stiffness. Biomaterials 2018, 178, 467-480. DOI: 10.1093/rb/rbx014. (36) Qi, Y. L.; Qi, H. P.; He, Y.; Lin, W. J.; Li, P. Z.; Qin, L.; Hu, Y. w.; Chen, L. P.; Liu, Q. S.; Sun, H. T.; Liu, Q.; Zhang, G.; Cui, S. Q.; Hu, J.; Yu, L.; Zhang, D. Y.; Ding, J. D., Strategy of Metal-polymer Composite Stent to Accelerate Biodegradation of Iron-based Biomaterials. ACS Appl. Mater. Interfaces 2018, 10 (1), 182-192. DOI: 10.1021/acsami.7b15206. (37) Ding, J. D., A Composite Strategy to Fabricate High-performance Biodegradable Stents for Tissue Regeneration. Sci. China Mater. 2018, 61 (8), 1132-1134. DOI: 10.1007/s40843-018-9228-4. (38) Pan, Z.; Ding, J. D., Poly(lactide-co-glycolide) Porous Scaffolds for Tissue Engineering and Regenerative Medicine. J. R. Soc.Interface Interface Focus 2012, 2 (3), 366-377. DOI: 10.1098/rsfs.2011.0123. (39) Wu, L. B.; Ding, J. D., In Vitro Degradation of Three-Dimensional Porous Poly(D,L-lactide-co-glycolide) Scaffolds for Tissue Engineering. Biomaterials 2004, 25 (27), 5821-5830. DOI: 10.1016/j.biomaterials.2004.01.038. (40) Caplan, A. I., What's in a Name? Tissue Eng., Part A 2010, 16 (8), 2415-2417. DOI: 10.1089/ten.tea.2010.0216. (41) Caplan, A. I., Mesenchymal Stem Cells: Time to Change the Name! Stem Cells Transl. Med. 2017, 6 (6), 1445-1451. DOI: 10.1002/sctm.17-0051. (42) Pittenger, M. F.; Mackay, A. M.; Beck, S. C.; Jaiswal, R. K.; Douglas, R.; Mosca, J. D.; Moorman, M. A.; Simonetti, D. W.; Craig, S.; Marshak, D. R., Multilineage Potential of Adult Human Mesenchymal Stem Cells. Science 1999, 284 (5411), 143-147. DOI: 10.1126/science.284.5411.143. (43) Lai, M.; Cai, K. Y.; Zhao, L.; Chen, X. Y.; Hou, Y. H.; Yang, Z. X., Surface Functionalization of TiO2 Nanotubes with Bone Morphogenetic Protein 2 and Its Synergistic Effect on the Differentiation of Mesenchymal Stem Cells. Biomacromolecules 2011, 12 (4), 1097-1105. DOI: 10.1021/bm1014365. (44) Wu, C.; Li, J. G.; Pang, P. F.; Liu, J. J.; Zhu, K. S.; Li, D.; Cheng, D.; Chen, J. W.; Shuai, X. T.; Shan, H., Polymeric Vector-Mediated Gene Transfection of MSCs for Dual Bioluminescent and MRI Tracking In Vivo. Biomaterials 2014, 35 (28), 8249-8260. DOI: 10.1016/j.biomaterials.2014.06.014. (45) Shen, T.; Dai, Y. K.; Li, X. G.; Xu, S. Z.; Gou, Z. R.; Gao, C. Y., Regeneration of the Osteochondral Defect by a Wollastonite and Macro-Porous Fibrin Biphasic Scaffold. ACS 37



Biomater. Sci. Eng. 2017. DOI: 10.1021/acsbiomaterials.7b00333. (46) Chen, J. Q.; Li, Y. J.; Wang, B.; Yang, J. B.; Heng, B. C.; Yang, Z.; Ge, Z. G.; Lin, J. H., TGF-β1 Affinity Peptides Incorporated within a Chitosan Sponge Scaffold Can Significantly Enhance Cartilage Regeneration. J. Mater. Chem. B 2018, 6, 675-687. DOI: 10.1039/c7tb02132a. (47) Zhou, K.; Ren, X. L.; Zhao, M. G.; Mei, X. F.; Zhang, P.; Chen, Z. H.; Zhu, X. D., Promoting Proliferation and Differentiation of BMSCs by Green Tea Polyphenols Functionalized Porous Calcium Phosphate. Reg. Biomater. 2018, 5 (1), 35-41. DOI: 10.1093/rb/rbx031. (48) Lewandrowski, K. U.; Bondre, S. P.; Wise, D. L.; Trantolo, D. J., Enhanced Bioactivity of A Poly(propylene fumarate) Bone Graft Substitute by Augmentation with Nano-Hydroxyapatite. Biomed. Mater. Eng. 2003, 13 (2), 115-124. (49) Bernards, M. T.; Qin, C. L.; Jiang, S. Y., MC3T3-E1 Cell Adhesion to Hydroxyapatite with Adsorbed Bone Sialoprotein, Bone Osteopontin, and Bovine Serum Albumin. Colloids Surf., B 2008, 64 (2), 236-247. DOI: 10.1016/j.colsurfb.2008.01.025. (50) Roohani-Esfahani, S. I.; Nouri-Khorasani, S.; Lu, Z. F.; Appleyard, R.; Zreiqat, H., The Influence Hydroxyapatite Nanoparticle Shape and Size on the Properties of Biphasic Calcium Phosphate Scaffolds Coated with Hydroxyapatite-PCL Composites. Biomaterials 2010, 31 (21), 5498-5509. DOI: 10.1016/j.biomaterials.2010.03.058. (51) He, Y.; Wang, X.; Chen, L.; Ding, J. D., Preparation of Hydroxyapatite Micropatterns for the Study of Cell-Biomaterial Interactions. J. Mater. Chem. B 2014, 2 (16), 2220-2227. DOI: 10.1039/c4tb00146j. (52) Agrawal, C. M.; Athanasiou, K. A., Technique to Control pH in Vicinity of Biodegrading PLA-PGA Implants. J. Biomed. Mater. Res. 1997, 38 (2), 105-114. DOI: 0021-9304/97/020105-10. (53) Shikinami, Y.; Okuno, M., Bioresorbable Devices Made of Forged Composites of Hydroxyapatite (HA) Particles and Poly-L-lactide (PLLA): Part I. Basic Characteristics. Biomaterials 2001, 22 (23), 3197-3211. DOI: 10.1016/S0142-9612(01)00072-2. (54) Olfert, E. D.; Mcwilliam, A. A.; Olfert, E. D.; Mcwilliam, A. A., Guide To The Care And Use Of Experimental Animals. Can. Vet. J. 1993, 26 (1), 49. DOI: 0-919087-18-3. (55) Pan, Z.; Qu, Z. H.; Zhang, Z.; Peng, R.; Yan, C.; Ding, J. D., Particle-Collision and Porogen-Leaching Technique to Fabricate Polymeric Porous Scaffolds with Microscale Roughness of Interior Surfaces. Chin. J. Polym. Sci. 2013, 31 (5), 737-747. DOI: 10.1007/s10118-013-1264-1. (56) He, Y.; Pan, Z.; Ding, J. D., Effects of Degradation Media of Polyester Porous Scaffolds on Viability and Osteogenic Differentiation of Mesenchymal Stem Cells. Acta Polym. Sin. 38


Page 38 of 45

Page 39 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


2013, 13 (6), 755-764. DOI: 10.3724/SP.J.1105.2013.12439. (57) Liang, X. Y.; Qi, Y. L.; Pan, Z.; He, Y.; Liu, X. N.; Cui, S. Q.; Ding, J. D., Design and Preparation of Quasi-Spherical Salt Particles as Water-Soluble Porogens to Fabricate Hydrophobic Porous Scaffolds for Tissue Engineering and Tissue Regeneration. Mater. Chem. Front. 2018, 2 (8), 1539-1553. DOI: 10.1039/c8qm00152a. (58) Qu, X.; Cui, W. J.; Yang, F.; Min, C. C.; Shen, H.; Bei, J. Z.; Wang, S. G., The Effect of Oxygen Plasma Pretreatment and Incubation in Modified Simulated Body Fluids on the Formation of Bone-Like Apatite on Poly(lactide-co-glycolide) (70/30). Biomaterials 2007, 28 (26), 9-18. DOI: 10.1016/j.biomaterials.2006.08.024. (59) Liu, W. Y.; Yeh, Y. C.; Lipner, J.; Xie, J. W.; Sung, H. W.; Thomopoulos, S.; Xia, Y. N., Enhancing the Stiffness of Electrospun Nanofiber Scaffolds with Controlled Surface Coating and Mineralization. Langmuir 2011, 27 (15), 9088-9093. DOI: 10.1021/la2018105. (60) Shi, G. X.; Wang, S. G.; Bei, J. Z., Preparation of Porous Cell Scaffolds of Poly(L-lactic acid) and Poly(L-lactic-co-glycolic acid) and the Measurement of Their Porosities. J. Funct. Polym. 2001, 14, 7-11. DOI: 10.3969/j.issn.1008-9357.2001.01.002. (61) Zhang, J. C.; Wu, L. B.; Jing, D. Y.; Ding, J. D., A Comparative Study of Porous Scaffolds with Cubic and Spherical Macropores. Polymer 2005, 46 (13), 4979-4985. DOI: 10.1016/j.polymer.2005.02.120. (62) Wu, L. B.; Zhang, J. C.; Jing, D. Y.; Ding, J. D., "Wet-state" Mechanical Properties of Three-dimensional Polyester Porous Scaffolds. J. Biomed. Mater. Res., Part A 2006, 76A (2), 264-271. DOI: 10.1002/jbm.a.30544. (63) Tang, J.; Peng, R.; Ding, J. D., The Regulation of Stem Cell Differentiation by Cell-Cell Contact on Micropatterned Material Surfaces. Biomaterials 2010, 31 (9), 2470-2476. DOI: 10.1016/j.biomaterials.2009.12.006. (64) Yan, C.; Sun, J. G.; Ding, J. D., Critical Areas of Cell Adhesion on Micropatterned Surfaces. Biomaterials 2011, 32 (16), 3931-3938. DOI: 10.1016/j.biomaterials.2011.01.078. (65) Peng, R.; Yao, X.; Ding, J. D., Effect of Cell Anisotropy on Differentiation of Stem Cells on Micropatterned Surfaces Through the Controlled Single Cell Adhesion. Biomaterials 2011, 32 (32), 8048-8057. DOI: 10.1016/j.biomaterials.2011.07.035. (66) Ye, K.; Cao, L. P.; Li, S. Y.; Yu, L.; Ding, J. D., Interplay of Matrix Stiffness and Cell-Cell Contact in Regulating Differentiation of Stem Cells. ACS Appl. Mater. Interfaces 2015, 8 (34), 21903-21913. DOI: 10.1021/acsami.5b09746. (67) Xu, K.; Chen, W. Z.; Mu, C. Y.; Yu, Y. L.; Cai, K. Y., Strontium Folic Acid Derivative Functionalized Titanium Surfaces for Enhanced Osteogenic Differentiation of Mesenchymal Stem Cells In Vitro and Bone Formation In Vivo. J. Mater. Chem. B 2017, 5 (33), 6811-6826. DOI: 10.1039/c7tb01529a. 39



(68) Cao, B.; Peng, Y. M.; Liu, X. N.; Ding, J. D., Effects of Functional Groups of Materials on Nonspecific Adhesion and Chondrogenic Induction of Mesenchymal Stem Cells on Free and Micropatterned Surfaces. Acs Appl. Mater. Interfaces 2017, 9 (28), 23574–23585. DOI: 10.1021/acsami.7b08339. (69) Tan, C. Y.; Singh, R.; Teh, Y. C.; Tan, Y. M.; Yap, B. K., The Effects of Calcium-to-Phosphorus Ratio on the Densification and Mechanical Properties of Hydroxyapatite Ceramic. Int. J. Appl. Ceram. Technol. 2015, 12 (1), 223-227. DOI: 10.1111/ijac.12249. (70) Long, G.; Zhong Yue, H.; Shi Feng, Y.; Kun Xi, Z.; Sheng Hua, X.; Gui Fei, L.; Lei, C.; Jing Bo, Y., Sr-HA-graft-poly(γ-benzyl-L-glutamate) Nanocomposite Microcarriers: Controllable Sr2+ Release for Accelerating Osteogenenisis and Bony Nonunion Repair. Biomacromolecules 2017, 18, 3742-3752. DOI: 10.1021/acs.biomac.7b01101. (71) Kay, M. I.; Young, R. A.; Posner, A. S., Crystal Structure of Hydroxyapatite. Nature 1964, 204 (4963), 1050-1052. DOI: 10.1038/2041050a0. (72) Langer, R.; Vacanti, J. P., Tissue Engineering. Science 1993, 260 (5110), 920-926. DOI: 10.1126/science.8493529. (73) Cao, Y.; Vacanti, J. P.; Paige, K. T.; Upton, J.; Vacanti, C. A., Transplantation of Chondrocytes Utilizing a Polymer-Cell Construct to Produce Tissue-Engineered Cartilage in the Shape of a Human Ear. Chin. J. Orthop. Trauma 1997, 100 (2), 297-302. DOI: 10.1097/00006534-199708000-00001. (74) Hutmacher; W., D., Scaffolds in Tissue Engineering Bone and Cartilage. Biomaterials 2000, 21 (24), 2529-2543. DOI: 10.1016/S0142-9612(00)00121-6. (75) Zhang, X. D.; Zou, P.; Wu, C.; Qu, Y.; Zhang, J. G., A Study of Porous Block HA Ceramics and Its Osteogenesis. Elsevier Applied Science: The Nether, Amsterdam, 1991; p 408-415. DOI: 10.1007/978-94-011-2896-4_57. (76) Ripamonti, U., Bone Induction in Nonhuman Primates. An Experimental Study on the Baboon. Clin. Orthop. Relat. Res. 1991, 269 (269), 284-294. DOI: 10.1097/00003086-199108000-00039. (77) Yamasaki, H.; Sakai, H., Osteogenic Response to Porous Hydroxyapatite Ceramics under the Skin of Dogs. Biomaterials 1992, 13 (5), 308-312. DOI: 10.1016/0142-9612(92)90054-R. (78) Toth, J. M.; Lynch, K. L.; Hackbarth, D. A., Ceramic-Induced Osteogenesis Following Subcutaneous Implantation of Calcium Phosphates. Bioceramics 1993, 6, 9-13. (79) Klein, C.; De, G. K.; Chen, W. Q.; Li, Y. B.; Zhang, X. D., Osseous Substance Formation Induced in Porous Calcium Phosphate Ceramics in Soft Tissues. Biomaterials 1994, 15 (1), 31-34. DOI: 10.1016/0142-9612(94)90193-7. 40


Page 40 of 45

Page 41 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(80) Ripamonti, U., Osteoinduction in Porous Hydroxyapatite Implanted in Heterotopic Sites of Different Animal Models. Biomaterials 1996, 17 (1), 31-35. DOI: 10.1016/0142-9612(96)80752-6. (81) Ripamonti, U.; Duneas, N., Tissue Engineering of Bone by Osteoinductive Biomaterials. MRS Bull. 1996, 21 (11), 36-39. DOI: 10.1557/S0883769400031833 (82) Yuan, H. P.; Yang, Z. J.; Li, Y. B.; Zhang, X. D.; Bruijn, J. D. D.; Groot, K. D., Osteoinduction by Calcium Phosphate Biomaterials. J. Mater. Sci.: Mater. Med. 1998, 9 (12), 723-726. DOI: 10.1023/A:1008950902047. (83) Yuan, H. P.; Kurashina, K.; de Bruijn, J. D.; Li, Y. B.; De, G. K.; Zhang, X. D., A Preliminary Study on Osteoinduction of Two Kinds of Calcium Phosphate Ceramics. Biomaterials 1999, 20 (19), 1799-1806. DOI: 10.1016/S0142-9612(99)00075-7. (84) Tang, Z.; Li, X.; Tan, Y.; Fan, H.; Zhang, X. D., The Material and Biological Characteristics of Osteoinductive Calcium Phosphate Ceramics. Regener. Biomater. 2018, 5 (1), 43-59. DOI: 10.1093/rb/rbx024. (85) Peppas, N. A.; Langer, R., New Challenges in Biomaterials. Science 1994, 263 (5154), 1715-1720. DOI: 10.1126/science.8134835. (86) Kim, B. S.; Baez, C. E.; Atala, A., Biomaterials for Tissue Engineering. World J. Urol. 2000, 18 (1), 2-9. DOI: 10.1007/s003450050002. (87) Shin, H.; Jo, S.; Mikos, A. G., Biomimetic Materials for Tissue Engineering. Adv. Drug Delivery Rev. 2003, 24 (2), 4353-4364. DOI: 10.1016/S0142-9612(03)00339-9. (88) Khademhosseini, A.; Langer, R., Microengineered Hydrogels for Tissue Engineering. Biomaterials 2007, 28 (34), 5087-5092. DOI: 10.1016/j.biomaterials.2007.07.021. (89) Place, E. S.; Evans, N. D.; Stevens, M. M., Complexity in Biomaterials for Tissue Engineering. Nat. Mater. 2009, 8 (6), 457-470. DOI: 10.1038/nmat2441. (90) Lutolf, M. P.; Gilbert, P. M.; Blau, H. M., Designing Materials to Direct Stem-Cell Fate. Nature 2011, 462 (7272), 433-441. DOI: 10.1038/nature08602. (91) Zhang, X. J.; Li, Y.; Chen, Y. E.; Chen, J. H.; Ma, P. X., Cell-free 3D Scaffold with Two-stage Delivery of MiRNA-26a to Regenerate Critical-sized Bone Defects. Nat. Commun. 2016, 7, 10376. DOI: 10.1038/ncomms10376. (92) Hollister, S. J., Porous Scaffold Design for Tissue Engineering. Nat. Mater. 2005, 4 (7), 518-524. DOI: 10.1038/nmat1421. (93) Jiang, C.-C.; Chiang, H.-S.; Lin, Y.-J.; Kuo, T.-F.; Shieh, C.-S.; Huang, Y.-Y.; Tuan, R. S., Repair of Porcine Articular Cartilage Defect with A Biphasic Osteochondral Composite. J. Orthop. Res. 2007, 25 (10), 1277-1290. DOI: 10.1002/jor.20442. 41



(94) Mano, J. F.; Silva, G. A.; Azevedo, H. S.; Malafaya, P. B.; Sousa, R. A.; Silva, S. S.; Boesel, L. F.; Oliveira, J. M.; Santos, T. C.; Marques, A. P., Natural Origin Biodegradable Systems in Tissue Engineering and Regenerative Medicine: Present Status and Some Moving Trends. J. R. Soc., Interface 2007, 4 (17), 999-1030. DOI: 10.1098/rsif.2007.0220. (95) Cao, L. P.; Li, Q. L.; Zhang, C.; Wu, H. C.; Yao, L. Q.; Xu, M. D.; Yu, L.; Ding, J. D., Safe and Efficient Colonic Endoscopic Submucosal Dissection Using an Injectable Hydrogel. ACS Biomater. Sci. Eng. 2016, 2 (3), 393−402. DOI: 10.1021/acsbiomaterials.5b00516. (96) Atluri, K.; De Jesus, A. M.; Chinnathambi, S.; Brouillette, M. J.; Martin, J. A.; Salem, A. K.; Sander, E. A., Blebbistatin-Loaded Poly(D,L-lactide-co-glycolide) Particles For Treating Arthrofibrosis. ACS Biomater. Sci. Eng. 2016, 2 (7), 1097-1107. DOI: 10.1021/acsbiomaterials.6b00082. (97) Barry, F.; Boynton, R. E.; Liu, B.; Murphy, J. M., Chondrogenic Differentiation of Mesenchymal Stem Cells from Bone Marrow: Differentiation-Dependent Gene Expression of Matrix Components. Exp. Cell Res. 2001, 268 (2), 189-200. DOI: 10.1006/excr.2001.5278. (98) Duan, X. H.; Lu, L. J.; Wang, Y.; Zhang, F.; Mao, J. J.; Cao, M. H.; Lin, B. L.; Zhang, X.; Shuai, X. T.; Shen, J., The Long-term Fate of Mesenchymal Stem Cells Labeled with Magnetic Resonance Imaging-Visible Polymersomes in Cerebral Ischemia. Int. J. Nanomed. 2017, 12, 6705-6719. DOI: 10.2147/IJN.S146742. (99) Buma, P.; Pieper, J. S.; Tienen, T. V.; Susante, J. L. C. V.; Kraan, P. M. V. D.; Veerkamp, J. H.; Berg, W. B. V. D.; Veth, R. P. H.; Kuppevelt, T. H. V., Cross-Linked Type I and Type II Collagenous Matrices for the Repair of Full-Thickness Articular Cartilage Defects—A Study in Rabbits. Biomaterials 2003, 24 (19), 3255-3263. DOI: 10.1016/S0142-9612(03)00143-1. (100) Nagura, I.; Fujioka, H.; Kokubu, T.; Makino, T.; Sumi, Y.; Kurosaka, M., Repair of Osteochondral Defects with A New Porous Synthetic Polymer Scaffold. J. Bone Jt. Surg. 2007, 89 (2), 258-264. DOI: 10.1302/0301-620X.89B2. (101) Liu, Z. Y.; Shi, W. S.; Ji, X. H.; Sun, C. S.; Jee, W. S.; Wu, Y. L.; Mao, Z. K.; Nagy, T. R.; Li, Q. N.; Cao, X., Molecules Mimicking Smad1 Interacting with Hox Stimulate Bone Formation. J. Biol. Chem. 2004, 279 (12), 11313-11319. DOI: 10.1074/jbc.M312731200. (102) Retting, K. N.; Song, B.; Yoon, B. S.; Lyons, K. M., BMP Canonical Smad Signaling Through Smad1 and Smad5 is Required for Endochondral Bone Formation. Development 2009, 136 (7), 1093-1104. DOI: 10.1242/dev.029926. (103) Skarżyńska, J.; Damulewicz, M.; Filipowska, J.; Madej, W.; Leboy, P. S.; Osyczka, A. M., Modification of Smad1 Linker Modulates BMP-Mediated Osteogenesis of Adult Human MSC. Connect. Tissue Res. 2011, 52 (5), 1-7. DOI: 10.3109/03008207.2010.551568. (104) Wang, W. G.; Song, B.; Teni, A.; Anna, S.; Sadik, J. E.; Lyons, K. M., Smad2 and Smad3 Regulate Chondrocyte Proliferation and Differentiation in the Growth Plate. PLoS Genet. 2016, 12 (10), e1006352. DOI: 10.1371/journal. 42


Page 42 of 45

Page 43 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(105) Fan, W. S.; Li, J. H.; Wang, Y. M.; Pan, J. F.; Li, S.; Zhu, L.; Guo, C. A.; Yan, Z. Q., CD105 Promotes Chondrogenesis of Synovium-Derived Mesenchymal Stem Cells Through Smad2 Signaling. Biochem. Biophys. Res. Commun. 2016, 474 (2), 338-344. DOI: 10.1016/j.bbrc.2016.04.101. (106) Ding, M.; Dalstra, M.; Linde, F.; Hvid, I., Mechanical Properties of the Normal Human Tibial Cartilage-Bone Complex in Relation to Age. Clin. Biomech. 1998, 13 (4–5), 351-358. DOI: 10.1016/S0268-0033(98)00067-9. (107) Yang, S.; Leong, K. F.; Du, Z.; Chua, C. K., The Design of Scaffolds for Use in Tissue Engineering. Part I. Traditional Factors. Tissue Eng., Part A 2002, 8 (1), 1-11. DOI: 10.1089/107632701753337645. (108) Jia, C.; Yu, D.; Lamarre, M.; Leopold, P. L.; Teng, Y. D.; Wang, H. J., Patterned Electrospun Nanofiber Matrices via Localized Dissolution: Potential for Guided Tissue Formation. Adv. Materi. 2014, 26 (48), 8192-8197. DOI: 10.1002/adma.201403509. (109) Li, S. Y.; Wang, X.; Cao, B.; Ye, K.; Li, Z. H.; Ding, J. D., Effects of Nanoscale Spatial Arrangement of Arginine-Glycine-Aspartate Peptides on Dedifferentiation of Chondrocytes. Nano Lett. 2015, 15 (11), 7755-7765. DOI: 10.1021/acs.nanolett.5b04043. (110) Wang, X.; Li, S. Y.; Yan, C.; Liu, P.; Ding, J. D., Fabrication of RGD Micro/Nanopattern and Corresponding Study of Stem Cell Differentiation. Nano Lett. 2015, 15 (3), 1457-1467. DOI: 10.1021/nl5049862. (111) Jia, C.; Luo, B. W.; Wang, H. Y.; Bian, Y. Q.; Li, X. Y.; Li, S. H.; Wang, H. J., Precise and Arbitrary Deposition of Biomolecules onto Biomimetic Fibrous Matrices for Spatially Controlled Cell Distribution and Functions. Adv. Mater. 2017, 29, 1701154. DOI: 10.1002/adma.201701154. (112) Tayton, E.; Purcell, M.; Aarvold, A.; Smith, J. O.; Briscoe, A.; Kanczler, J. M.; Shakesheff, K. M.; Howdle, S. M.; Dunlop, D. G.; Oreffo, R. O., A Comparison of Polymer and Polymer-Hydroxyapatite Composite Tissue Engineered Scaffolds for Use in Bone Regeneration. An In Vitro and In Vivo Study. J. Biomed. Mater. Res., Part A 2014, 102 (8), 2613-2624. DOI: 10.1002/jbm.a.34926. (113) Hunziker, E. B.; Rosenberg, L. C., Repair of Partial-Thickness Defects in Articular Cartilage: Cell Recruitment from the Synovial Membrane. J. Bone Jt. Surg., Am. Vol. 1996, 78 (5), 721-733. DOI: 10.2106/00004623-199605000-00012. (114) Kramer, J.; Böhrnsen, F.; Lindner, U.; Behrens, P.; Schlenke, P.; Rohwedel, J., In Vivo Matrix-Guided Human Mesenchymal Stem Cells. Cell Mol. Life Sci. 2006, 63 (5), 616-626. DOI: 10.1007/s00018-005-5527-z. (115) Chang, H. L.; Cook, J. L.; Mendelson, A.; Moioli, E. K.; Yao, H.; Mao, J. J., Regeneration of the Articular Surface of the Rabbit Synovial Joint by Cell Homing: a Proof of Concept Study. Lancet 2010, 376 (9739), 440-448. DOI: 43



10.1016/S0140-6736(10)60668-X. (116) Chen, P. F.; Tao, J. D.; Zhu, S. A.; Cai, Y. Z.; Mao, Q. J.; Yu, D. S.; Dai, J.; Ouyang, H. W., Radially Oriented Collagen Scaffold with SDF-1 Promotes Osteochondral Repair by Facilitating Cell Homing. Biomaterials 2015, 39, 114-123. DOI: 10.1016/j.biomaterials.2014.10.049. (117) Leach, J. K.; Whitehead, J. R., Materials-directed Differentiation of Mesenchymal Stem Cells for Tissue Engineering and Regeneration. ACS Biomater. Sci. Eng. 2018, 4, 1115−1127. DOI: 10.1021/acsbiomaterials.6b00741. (118) Bos, R. R.; Rozema, F. R.; Boering, G.; Nijenhuis, A. J.; Pennings, A. J.; Verwey, A. B.; Nieuwenhuis, P.; Jansen, H. W., Degradation of and Tissue Reaction to Biodegradable Poly(L-lactide) for Use As Internal Fixation of Fractures: a Study in Rats. Biomaterials 1991, 12 (1), 32-36. DOI: 10.1016/0142-9612(91)90128-W. (119) Bergsma, J. E.; de Bruijn, W. C.; Rozema, F. R.; Bos, R. R.; Boering, G., Late Degradation Tissue Response to Poly(L-lactide) Bone Plates and Screws. Biomaterials 1995, 16 (1), 25-31. DOI: 10.1016/0142-9612(95)91092-D. (120) Filardo, G.; Kon, E.; Perdisa, F.; Sessa, A.; Martino, A. D.; Busacca, M.; Zaffagnini, S.; Marcacci, M., Polyurethane-based Cell-free Scaffold for the Treatment of Painful Partial Meniscus Loss. Knee Surg. Sport. Tr. A. 2016, 25 (2), 1-9. DOI: 10.1007/s00167-016-4219-6. (121) Buma, P.; Schreurs, W.; Verdonschot, N., Skeletal Tissue Engineering-from In Vitro Studies to Large Animal Models. Biomaterials 2004, 25 (9), 1487-1495. DOI: 10.1016/S0142-9612(03)00492-7. (122) Chu, C. R.; Szczodry, M.; Bruno, S., Animal Models for Cartilage Regeneration and Repair. Tissue Eng., Part B 2010, 16 (1), 105-115. DOI: 10.1089=ten.teb.2009.0452. (123) Li, J. J.; Kaplan, D. L.; Zreiqat, H., Scaffold-Based Regeneration of Skeletal Tissues to Meet Clinical Challenges. J. Mater. Chem. B 2014, 2 (42), 7272-7306. DOI: 10.1039/c4tb01073f.

44


Page 44 of 45

Page 45 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


For Table of Contents use only

Graphical Abstract

Bilayered PLGA/PLGA-HAp Composite Scaffold for Osteochondral Tissue Engineering and Tissue Regeneration Xiangyu Liang1,†, Pingguo Duan2,†, Jingming Gao1, Runsheng Guo2, Zehua Qu1, Xiaofeng Li2, Yao He1, Haoqun Yao2, Jiandong Ding1,*

A bilayered

polymeric/inorganic

porous

compression-molding/porogen-leaching

scaffold

with

was

plasma-treated

developed surface

by

combining

deposition

for

osteochondral repair. Both in vitro and in vivo experiments were carried out. The in situ osteochondral regeneration potential of the scaffolds was confirmed via biomechanics analysis, histological evaluations and Western blot etc.

45


PLGA-HAp Composite Scaffold for Osteochondral

Recommend Documents