Injectable and thermo-sensitive hydrogel and PDLLA electrospun

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Injectable and thermo-sensitive hydrogel and PDLLA electrospun nanofiber membrane composites for guided spinal fusion Ying Qu, BeiYu Wang, Bingyang Chu, Chenlu Liu, Xin Rong, Hua Chen, JinRong Peng, and Zhiyong Qian ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b17020 • Publication Date (Web): 16 Jan 2018 Downloaded from http://pubs.acs.org on January 16, 2018

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ACS Applied Materials & Interfaces

Injectable and thermo-sensitive hydrogel and PDLLA electrospun nanofiber membrane composites for guided spinal fusion Ying Qu1, BeiYu Wang1, BingYang Chu1, ChenLu Liu1, Xin Rong1, Hua Chen1, JinRong Peng1, ZhiYong Qian1 * 1

Department of Hematology and Research Laboratory of Hematology, State Key Laboratory of

Biotherapy, West China Hospital, Sichuan University and Collaborative Innovation Center, Sichuan, China

Abstract Spinal fusion is the classic treatment to achieve spinal stability for the treatment of the spinal disease. Generally, spinal fusion still has to combine a certain of bone matrix for promoting bone formation to achieve the desired fusion effect based on the surgery, including the traditional bone matrix, such as the autologous bone, allografts and xenografts. Nevertheless, some problems still existed such as the immunogenic problems, the secondary wound, and pathogenic transfer and so on. Here the injectable thermosensitive hydrogel could substitute to avoid the problems as a potential biological scaffold for tissue engineering. Once injected, they could fill in the irregular-shaped cavity and change to a gel state at physiological temperature. Then we would like to design the collagen/n-HA/BMP-2@PCEC/PECE hydrogel composites based on previous work about collagen/n-HA/PECE hydrogel for exhibit better performance in guiding spinal fusion due to the addition of BMP-2@PCEC nanoparticles (PCEC, PCL-PEG-PCL). However, when the hydrogels were injected, one of the surfaces was in contact with the spine, but others were with soft tissue like muscles and fascia. The release behavior was the same at the different surfaces, so the factors could be released into the soft tissue, then it may be consumed or lead to ectopic bone formation. The hydrogel composites should be improved to adjust the direction of the releaser behavior. In consequence, we wrapped an electrostatic spinning nanofiber membrane possessing hydrophobicity around the hydrogels. In this study we developed a system that the collagen/n-HA/BMP-2@PCEC/PECE hydrogels were wrapped with the hydrophobicity PDLLA electrospun nanofiber membrane, setting up a barrier between the hydrogels and the soft tissue. The system could exhibit the biocompatibility, prevent the factors from escaping to keep their retention in the needed places of osteogenesis and the results demonstrated that it exhibited the excellent effect on the spinal fusion. KEYWORDS spinal fusion, bone regeneration, thermo-sensitive hydrogel, electrospun nanofiber membrane, BMP-2 *

To whom should be corresponded, Tel/Fax: +86-28-85501986, E-mail: [email protected] (Qian ZY).

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Introduction Bone is an important organ of the human body, with the support, protection, hematopoietic, calcium storage, metabolism and other functions, which itself has the ability to regenerate and self-repair.1, 2 And the spine is an important component of the bone that connects the skull to the pelvis of the human body. It is equipped with the following basic functions: supporting the head and torso, controlling the movement of the body and protecting the spinal cord and part of the nerve3. Spinal disease is a common type of skeletal system disease that seriously affects human health and quality of life. The surgical reconstruction of spinal stability is one of the important goals of spine surgery and spinal fusion is a classic treatment to achieve spinal stability whose principle was to stabilize two or more parts of the spine for integration, while the rest of the segment can move normally without affecting the normal physiological function of the spine, thus reducing the patient’s pain caused by the instability of spine4-7. Generally, spinal fusion still has to combine with a certain of bone matrix for promoting bone formation to achieve the desired fusion effect 8. At present, the common bone matrix promoting bone formation includes the autologous bone, allogeneic bone, xenograft and artificial bone9, 10. But there are still many problems. For example, the autologous bone has a good ability to promote osteogenesis as the “gold standard”11, while the limited quantity is unable to meet the needs of osteogenesis and the patients have to undergo a second operation increasing the pain. As for the xenograft and allogeneic bone, the potential danger still exists such as a certain degree of immunogenicity, and disease transmission.11,

12

In the long-term

scientific researches, many metal materials, ceramics and synthetic polymer materials were used to repair bone defects13, 14. However, some problems still could not be avoided such as toxicity, autoimmune rejection and difficult biodegradation that can’t meet the clinical demand. Therefore, it has drawn greater attention about the successful preparation of the biocompatible composite materials for spinal fusion.


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In the field of biomaterials, hydrogels have been widely used in tissue engineering on account of the injectable properties and their excellent ability to load growth factors or engineering cells.15-17 The thermo-sensitive hydrogel has good mobility at the room temperature and could change to the gel state at the physiological temperature in vovo.18-20 Due to its mobility, the hydrogel could contact fully with the bone in situ especially for the irregular region like the spine, thus it could solve the problem that the matrix promoting the bone regeneration could not fit the surface of the bone fully.21, 22 In our previous reports23, we developed a hydrogel using the poly(ethylene glycol)-poly(ε-caprolactone)-poly(ethylene

glycol)

copolymer

(PEG-PCL-PEG,

PECE) with the above-mentioned thermo-sensitivity as well as biodegradation, low toxicity, and biocompatibility and so on. To mimic the component of the bone that was consist of about 30% organic matter and 70% inorganic matter, we loaded nano-hydroxyapatite (n-HA) and collagen into the PECE hydrogel and prepared the injectable and thermos-sensitivity collagen/n-HA/PECE hydrogel composite for the skull regeneration. BMP was one of the growth factors that plays an important role in osteogenesis, which could make not only undifferentiated mesenchymal cells recruit to the bone formation center but also differentiate into bone cells in the early stage of bone formation.3, 24, 25 The exogenous BMPs was very easy to be degraded, assimilated and diluted by the body fluids.26According to the reports, while the protein was loaded into the nanoparticle, its uptake by cells and the release time could be improved.27, 28 Then in order to improve the osteogenic effect for spinal fusion, we planned to add the BMP-2(Bone Morphogenetic Protein-2) growth factors nanoparticles into the abovementioned collagen/n-HA/PECE composite to make the BMP-2 release sustainably. The hydrogels composites could reconstruct the microstructure of bone tissue to promote bone regeneration and control the release of the BMP-2 to adjust the osteogenesis microenvironment, while the release behavior was the same at the different surfaces. When the hydrogels were injected, one of the surfaces was in contact with the spine, but the others were with soft tissue like muscles and fascia. ACS Paragon Plus Environment


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The factors could be released into the soft tissue, then they may be consumed or lead to ectopic bone formation. All the time the balance of BMP-2 retention within the bone defect and BMP-2 release into surrounding soft tissues is very important.26 So the hydrogel composites should be improved to adjust the direction of the release behavior. The electrospun nanofiber membrane was an ideal cells scaffold that the morphology is similar to extracellular matrix.29,

30

The diameter of the fiber, hydrophilic and

hydrophobic properties and other characteristics could be adjusted through a series of parameters.31 Due to the hydrophobicity of the PDLLA, we developed a system that the hydrogels were wrapped with the PDLLA-based electrospun nanofiber membrane. To prevent the factors from escaping and make them retained in the needed places of osteogenesis. In conclusion, the goal of this work was to investigate the performance of the spinal fusion with the nanofiber membrane as a barrier to water-soluble macromolecules with the soft tissue.

EXPERIMENTAL SECTION Materials We

purchased

poly(ethylene

glycol)

methyl

ether

(MPEG,

Mw=550,

Sigma-Aldrich, USA), ε-caprolactone (ε-CL, Sigma-Aldrich, USA), Poly(ethylene glycol) (PEG, Mn= 4000, Sigma-Aldrich, USA), Tin(II) 2-etheylhexanoate (Sn(Oct)2, Sigma-Aldrich, USA), Hexamethylene diisocyanate (HMDI, Aldrich, USA), Sodium Dodecyl Sulfonate (SDS, Sigma-Aldrich, USA), Poly(D, L-lactide) (PDLLA, Mw=75000~120000, Aldrich), nano-hydroxyapatite (n-HA, Aladdin, China), recombinant human BMP-2 (rhBMP-2, Beijing Wishbiotechnology CO.,LTD, China), BMP-2 Elisa Kit (Neobioscience CO.,LTD, China). Synthesis of poly (ε-caprolactone)-PEG-poly(ε-caprolactone) copolymer PCEC and preparation of PCEC nanoparticle, BMP-2@PCEC nanoparticle


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PCEC copolymer was synthesized by ring-opening copolymerization as our previous report27,28. Typically, the main procedures were as follows: 4 g of PEG (Mn= 4000) and 96 g ε-caprolactone was introduced into a dry three-necked glass flask under nitrogen atmosphere. After residual moisture of PEG and ε-caprolactone was removed, several drops of Sn(Oct)2 was added. The mixtures were kept at 130oC and stirred for 6 h. Then the system was heated to 180 oC under vacuum condition and kept for another 30 min. While at this time the system was cooled to the room temperature under nitrogen atmosphere, the liquid was dissolved into the methylene chloride and reprecipitated from the filtrate using excess cold petroleum ether. At last the PCEC copolymer was filtered and vacuum dried to constant weight. We get the purified PCEC copolymer. Then the structure and the molecular were examined by 1H nuclear magnetic resonance spectroscopy (1H-NMR) and the Gel Permeation Chromatography spectrum(GPC). The anionic nanoparticles were prepared by modified emulsion solvent evaporation previously method27. 30mg PCEC was dissolved in 5ml acetone-ethyl acetate solvent and 5mg SDS was in 10ml water. First the SDS solution was emulsified under stirring with a rotor-stator device (T18, IKA, Germany) at speed of about 10,000 rpm. About sixteen minutes later, the O/W emulsion formed and it was evaporated by rotor evaporation immediately at 37oC, 100 rpm and -0.08mPa. At last, the slurry was dialyzed to remove the free SDS. Then we got the anionic PCEC nanoparticles. 100µl different concentration of BMP-2 solution was dropped into 500µl PCEC nanoparticle that the solid concentration of the result solution was adjusted to 3 mg/ml under slight magnetic stirring at 0oC for 30 min. The particle size distribution and zeta potential of nanoparticles were determined by dynamic light scattering laser particle size analyzer (DLS, Malvern Nano-ZS 90) at 25oC. The morphological characteristics were examined by transmission electron microscopy (TEM, H-6009IV, Hitachi, Japan).



Preparation of Collagen/n-HA/BMP-2 @PCEC/PECE hydrogel composite The triblock copolymer PEG-poly (ε-caprolactone)-PEG was synthesized in two steps according to our previous reports18. Firstly, MPEG-PCL copolymer was prepared by ring-opening copolymerization of ε-CL initiated by MPEG using Sn(Oct)2 as catalyst. After that the MPEG-PCL was cross-linked by using HMDI as linker. Then the structure of the copolymer was characterized by 1H-NMR. 1.2g PECE copolymer was dissolved in normal saline to prepare a solution with concentration of 30% (w/v). Then the solution was incubated in the water bath at 60oC for a few minutes, after that it was put into the ice-bath for another few minutes until the solution turned into a transparent liquid. From the above, the blank PECE sol was got. Next it was the preparation of the hydrogel composite. 0.2g collagen and 0.6g n-HA was added into the blank sol and the mixture was stirred and ultrasonicated for a while to make sure that they were dispersible uniformity in the sol. 200µl PCEC nanoparticle loading BMP-2 was added into the above solution and dispersible uniformity. Characterization of collagen/n-HA/BMP-2@PCEC/PECE hydrogel composite Scanning electron microscope (SEM) was used to investigate the morphology of the hydrogel composite. The samples were lyophilized, then were cross sectioned and sputtered with gold before observation with a JEOL SEM (JSM-5900LV, JEOL, Japan) Rheological measurement was carried out to investigate the sol-gel behavior of the hydrogel composite using an HAAKE Rheostress 6000 rheometer (Thermo scientific, USA) using parallel plates. The collagen/n-HA/BMP-2@PCEC/PECE hydrogel composites were incubated in the ice bath for a few minutes, then the samples were placed between parallel plates of 20 mm diameter and with a gap of 1 mm. The heating rate of the temperature was 1oC/min from 4oC to 60oC. The storage modulus (G’) and the loss modulus (G’ ’) was measured during the period under a controlled


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stress of 4.0 dyn/cm2 and a frequency of 1.0 Hz. The time of gelation was confirmed by observing the changing of the modulus. Fabrication of the PDLLA electrospun membranes Firstly, the electrospun solution at the concentration of 10%(w/v, PDLLA/CH2Cl2) was prepared. Then the electrospun membrane was prepared at a high voltage of 18 Kv (electrospinning apparatus, High Voltage Technology Institute, Beijing, China). The flow rate was adjusted to 6ml/h through a syringe pump and the distance between the nozzle and the collector was about 10 cm. The membrane that produced were dried in a vacuum oven at 40oC overnight to eliminate residual solvent. In vitro release behavior of BMP-2 500µl hydrogel composite containing 5µg BMP-2 was placed into the 6 of 5 mL-Eppendorf (EP) tubes to gel in an incubator at 37oC for 12 h. Among them, in the 3 of the tubes, PDLLA electrospun membranes were covered on the surfaces of the gels. Then the gels were immersed in 2ml preheating PBS (pH 7.4) and shaken at 100 rpm at 37oC. At determined time, half of the release media were collected and replaced with the same volume pre-heating fresh media. Then the release media were centrifuged at 13000 rpm and the supernatants were stored at -20oC until analysis. The concentration of BMP-2 was measured with the Elisa Kit. Degradation study of the hydrogel in vivo In vivo biocompatibility of the hydrogel and PDLLA electrospun membrane composite was investigated by implanting the materials into the subcutaneous tissue of SD rats. The experiments were approved by the Institutional Animal Care and Use Committee and were in compliance with all regulatory guidelines. An incision was made at the dorsal sites and the composite was implanted. After that, the incision was sewed up respectively. Then, the rats were sacrificed at predetermined intervals post-surgery (7days, 14days, 28days, 42days). The materials implanted and the



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surrounding tissues were harvest and analyzed by histology. Hematoxylin and eosin (HE) staining was used to study the biocompatibility. Surgery and Animal Care The experiments in vovo were on the New Zealand rabbits weighing range from 3.5 to 4 kg. The study design was based on posterolateral spine fusion in rabbit model, which was approved by the Institutional Animal Care and Use Committee and was in compliance with all regulatory guidelines. Before the operation the animals were anesthetized by intravenous injection of pentobarbital sodium provided by West China Hospital, Sichuan University. The hair of the surgical area was clipped and sterilized with iodine solution and medicinal alcohol. Then an incision was made at dorsal midline skin followed by two paramedian fascial incisions to expose the L4 L5 spine or L5- L6 with the electric scalpel. Decortication of the spine was performed by high-speed bur. Then the composites were planted into the areas and the fascia and skin were sewed up separately with absorbable sutures (Fig 6 was the photo of the surgical procedure). One of the four groups (group a) was injected 4ml collagen/n-HA/PECE hydrogel composite, group b was 4ml collagen/n-HA/ PECE hydrogel composite while the three surfaces that contacted the muscles were coated with

the

PDLLA

electrospun

membrane,

group

c

was

injected

collagen/n-HA/BMP-2@PCEC/PECE hydrogel composite and group d was collagen/n-HA/BMP-2@PCEC/PECE hydrogel composite coated with PDLLA electrospun membrane (10µg BMP-2 was loaded into the 4ml hydrogel system). After surgery, the rabbits received 400,000 units penicillin per day immediately for 3 days. Then at 4 weeks, 12 weeks and 20 weeks post-surgery, the animals were sacrificed for the evaluation of the bone regeneration. Evaluation of bone regeneration capacity Radiological examination


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At determined time, the rabbits were anesthetized and radiographed to assess the new bone formation and the fusion using a digital a digital X-ray unit (5s exposure at 62 kVP and 250mA). Micro CT Examination

The rabbits were sacrificed with an overdose of sodium pentobarbital at 4 weeks, 12 weeks and 20 weeks post-surgery. The harvested operative sites of the spines were scanned using Micro-computational tomography (Micro-CT) scanner (X-ray voltage = 55 kV, X-ray current = 90 µA, and voxel resolution = 20 µm). The dates were then reconstructed to create 3D geometry using VGStudioMax. Histological analysis

At determined time, the animals were sacrificed and the harvested samples were fixed with 10% neutral formalin. Then the fixed bony specimens were decalcified in 12.5% EDTA for 6 weeks at 37 oC and shaken at 100 rpm for complete decalcification and the sequential water substitution process was proceeded. After that, they are all embedded in paraffin wax. Serial longitudinal and cross sections with 5 µm thickness were made using a microtome and stained for H&E, Trichrome-Masson according to standard protocols. Results and discussion Evaluation of obtained nanoparticles Synthesis and Characterization of PCEC Copolymer

PCEC copolymer was synthesized by ring-opening copolymerization. According to the result of the 1H-NMR, the peak 3.65 ppm (e) was assigned methylene protons of PEG units (-CH2CH2O-), the peak 2.32 ppm and 4.07 ppm were attributed to methylene protons of -OCCH2- and -CH2OOC- in PCL units and 1.32 ppm and1.56 ppm were repetitive methylene protons peak of PCL. So we prepared PCEC copolymer successfully (Fig 1A). From the GPC spectrum of PCEC copolymer, we



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found that it was a single peak (Fig 1B). It indicated that the macromolecular weight was the uniform distribution and there was no other byproducts, the molecular weight was 46806. Then we prepared the PCEC nanoparticle. According to the previous reports27, it was prepared by emulsion solvent evaporation method. Because BMP-2 is a kind of cationic protein, we need to prepare the anionic nanoparticle and choose the SDS as the emulsifying agent. BMP-2 was loaded on the surface of the anionic PCEC nanoparticles due to electrostatic interactions.

The size

of blank nanoparticles was 90 nm and the zeta potential was -29.13 mV. Then 10µg, 15µg and 20µg BMP-2 protein was added into 3mg anionic PCEC nanoparticles solutions. The particle size distribution and the TEM photo of the BMP-2@PCEC nanoparticles were showed in the Fig 2 and we could find that the size distribution turned a little wider. The size and the zeta potential of nanoparticle loading BMP-2 did not change obviously compared with the blank PCEC nanoparticles. The BMP-2 level of the supernatant was measured by the Micro-BCA Protein Assay Kit after a highly speed centrifugation. PCEC was much more than BMP-2 protein, so the adsorption efficiencies may achieve almost 100%. And the BMP-2@PCEC nanoparticles were homogeneous and steady during the needed period. Characterization of PECE/Collagen/n-HA / BMP-2@PCEC hydrogel composite From the characteristic peak of the 1H-NMR result, we could judge that we synthesized the PECE copolymer successfully (Fig 1C). The peak of 3.38 ppm was assigned methylene protons of PEG units (-CH2CH2O-), then the weak peak of 4.22 ppm(c) and 3.70 ppm(d) was attributed the protons of -OCH2CH2 between the MPEG and PCL unites. The two peaks at the same intensity of 2.30(e) ppm and 4.10 ppm(g) are the methylene proton characteristic absorption peaks in the PCL block. The absorption peaks (h, i, j) represent proton absorption peak on the methylene of the HMDI crosslinker.


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PECE was a kind of thermosensitive hydrogel and our group have carried out a lot of researches18,19,21,23. It was injectable and thermo-sensitive, which means it could flow freely at room temperature and would change to gel state at 37 oC. According to the previous reports23, we prepared the collagen/n-HA/PECE hydrogel composite for imitating bone matrix components and the hydrogel composite exhibited the similar thermo-sensitivity. In this study, the BMP-2@PCEC nanoparticles were added in the collagen/n-HA/PECE hydrogel composite. The sol-gel behavior could be confirmed by the rheology. With the temperature increasing, the storage modulus (G’) and loss modulus (G”) were monitoring by the Hakke rheometer. In the Fig 3A, the G’ and G’’ of the hydrogel composite were low when the temperature was below 33oC. It reveals the composite still could flow. When the temperature reached above 33oC, the G’ and G’’ increased observably and the G’ value was higher than G’’, it demonstrated that the composite had been in a gel state. With the further increase of temperature, the G’ and G’’ decreased obviously, it demonstrated that the composites changed to be in a flow state. Based on the above-mentioned results, the hydrogel composite had obvious thermo-sensitive after the addition of the PCEC nanoparticles compared the previous report about the collagen/n-HA/PECE hydrogel composite23. Then we observed the inner structure of the collagen/n-HA/BMP-2@PCEC/PECE hydrogel composite by scanning electron microscopy(SEM). The internal network porous structure was showed in Fig 3B and the pores were connected. The hydrogel composite could store water due to the exits of the network porous structure. And it was conducive to the transmission and transport of nutrients, providing a favorable stent conditions for the growth of cells in bone regeneration. The addition of PCEC nanoparticles did not affect the structure of the hydrogel composite compared with collagen/n-HA/PECE hydrogel composite. It showed the inner-structure of the PDLLA electrospun membrane in Fig 4A, and the diameter of the fiber was 10µm. The fiber surface is relatively smooth with a small pore structure. In vitro release behavior of BMP-2



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According to the Fig 4B, BMP-2 could be released from the hydrogel composite over a sustained period. The cumulative release rate at 180h was 30.6%. While the release rate of the composite that was wrapped with the PDLLA electrospun membrane was lower too much. At the same time, the rate was 16%. It means that the BMP-2 could be released continuously, but the membrane can prevent it from escaping. It is to say that the membrane could decrease the loss of the BMP-2 to make more keep in the places needed. Degradation study of the hydrogel in vivo We planted the composite into the back of the rats and with the time increasing, the hydrogels gradually decreased and completely degraded in the end. In the period, a thin coating on the surface of the system was formed and the large amount of angiogenesis was showed. It was proved that this hydrogel and PDLLA electrospun membrane composite exhibited good biocompatibility. In Fig 5, it was showed that the system began to decrease gradually on the 14th days, and disappeared substantially on the 42th days. It could be demonstrated by the HE staining that obvious inflammatory cells could be seen on the 7th days after implantation and inflammatory reaction gradually disappeared after 42 days. In addition, there was no any obvious suppuration, hematoma and other similar phenomena in the whole process of degradation.

In

summary,

the

system

revealed

good

biodegradable

and

biocompatibility. In previous report, the collagen/n-HA/PECE hydrogel was biodegradable and biocompatible and the system with the addition of the PDLLA electrostatic spinning nanofiber membrane was still the same. Evaluation of bone defect treatment Radiological analysis We investigated the regeneration of the different groups by the imaging analysis including Micro-CT and X-ray. Among the groups, group a was collagen/n-HA/PECE hydrogel composite, group b was collagen/n-HA/PECE hydrogel composite coated with

PDLLA

electrospun

membrane,


group

c

was

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collagen/n-HA/BMP-2@PCEC/PECE hydrogel composite, and group d was collagen/n-HA/BMP-2@PCEC/PECE hydrogel composite coated with PDLLA electrospun membrane. At 20th weeks, we preliminary evaluated the bone regeneration effect. It demonstrated a certain amount of new bone formation along the edge of the spine in all groups. The hydrogel system was coated with the PDLLA electrospun membrane in group b, and the membrane prevented the collagen and n-HA loaded in the hydrogel from escaping. The osteogenesis effect of group b was enhanced and new bone mass was more than group a. The BMP-2@PCEC nanoparticles were added in the group c compared with group a.BMP-2 is a kind of exogenous growth factor which could induce the osteoblast differentiation of mesenchymal cells and promote osteogenesis in vivo. It was showed in the Fig7 that the shadow area of new bone was larger, and that was to say that the bone mass was significantly increased. On that basis, the density was increased too, which means that the new bone remodel better. The BMP-2@PCEC nanoparticles was added and the PDLLA electrospun membrane was coating in the group d. It exhibited that the bone mass was obviously more than the other three groups, the density of shadow was greater and the density of new tissue was higher. In conclusion, the collagen/n-HA/BMP-2@PCEC/PECE hydrogel composite coated with PDLLA electrospun membrane exhibited the most excellent effect of bone regeneration. Then we evaluated the bone regeneration by the cross section and vertical plane of Micro-CT (Fig 8). Four weeks after surgery, some new bone was generated along the edge of the polished parts. The mass about d group was more than the other groups. With the time increasing, the new bone mass was also increased and the structure of bone trabecula was generated. Simultaneously, the interface between the host bone and the new bone could not be distinguished and more regular trabecular bone morphology could be seen. But the interfaces could be distinguished clearly in others. The most important was that system had guided the spinal fusion. It demonstrated that the system of collagen/n-HA/BMP-2@PCEC/PECE hydrogel composite coated with



PDLLA electrospun membrane exhibited excellent ability in guiding bone regeneration. These results showed that the incorporation of BMP-2@PCEC nanoparticles with the collagen/n-HA/PECE hydrogel could promote the bone regeneration. The exogenous BMPs was very easy to assimilated and diluted by the body fluid, thus loses its function. The hydrogel composite could make it release slowly in local as an idea carrier. While the release behavior was the same at different surfaces including the surfaces in contact with the soft tissues. It was very easy to release to the soft tissues and escape for BMP-2 and other factor or nutrient, so there were no enough factors to play their role effectively in the needed place. The membrane prevented them from escaping and made them more retainable. The effect of bone regeneration could be enhanced. Histological evaluation At predetermined time points, the surgical site was fixed and decalcified, then the histomorphological analysis was performed (Fig 9A and B). At 4 weeks after surgery, a certain amount of bone matrix with a lot of collagen was generated along the host bone in all the groups. While a large number of new bony structure has appeared in the group d. The composite system has been already in degradation progress so that they could provide the growth space for the new bone. During degradation, collagen exposure was more favorable for cell growth, more conducive for cell adhesion and derivatization. After 12 weeks, a small amount of blood vessels has been generated which provided a wealth of blood supply and nutrients for bone regeneration in group b. Then a lot of newly formed collagen was at the edge of the host bone in group a and b. a large number of bone-like tissue has grown in group c, even mature bone marrow and trabecular bone structure has formed in group d. After another 8 weeks, some bone marrow has formed in group a and b, while mature and stable trabecular bone structure generated. It was the overall histologic images and the partial magnification in Fig 9C and it was obvious that new bone mass was much more in group d than other three groups. There is a lot of collagen deposition not yet bony in the three groups, but the group d ACS Paragon Plus Environment

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was the opposite, almost the most has been completely bony. There were already no obvious boundaries between the new bone and host bone while mature and stable trabecular bone structure and mature bone marrow were formed.

Conclusion In

summary,

we

developed

collagen/n-HA/BMP-2@PCEC/PECE

a

hydrogel

system composite

combining with

the

the PDLLA

electrospun nanofiber membrane. The hydrogels were wrapped with the hydrophobicity PDLLA electrospun nanofiber membrane, setting up a barrier between the hydrogels and the soft tissue. The system could prevent the factors from escaping to keep their retention in the needed places of osteogenesis and the results demonstrated that it exhibited the excellent effect on spinal fusion. Acknowledgement This work was financially supported by The National Key Research and Development Program of China (2017YFC1103500, 2017YFC1103502), the National Natural Science Foundation (31525009, 31271021), Distinguished Young Scholars of Sichuan University (2011SCU04B18), and Sichuan Innovative Research Team Program for Young Scientists (2016TD0004).

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Figure Captions Scheme1 Schematic illustrating about the thermo-sensitive hydrogel and PDLLA electrospun nanofiber membrane composites for the spinal fusion. Figure1 A. Structure and 1H-NMR spectrum of PCEC copolymer; B. The GPC spectrum of PCEC copolymer; C. Structure and 1H-NMR spectrum of PECE copolymer. Figure2 A. The particle size distributions of prepared blank and BMP-2 loaded PCEC nanoparticles. The size of blank nanoparticles was 90 nm and the zeta potential was -29.13 mV. The size distribution turned a little wider after BMP-2 was loaded into the blank nanoparticles. The size and the zeta potential of nanoparticle loading BMP-2 did not change obviously compared with the blank PCEC nanoparticles; B. The TEM photo of the PCEC nanoparticles loading the BMP-2. Figure3 A. The rheological characteristics of the hydrogel composite. The hydrogel composites turned to a gel state from a flow state while the G’ value was higher than G’’ at 33oC. The hydrogel composite had obvious thermo-sensitive; B. The morphologic photo of the hydrogel composite. The hydrogel composite owned the connected network porous structure. Figure4 A. The morphologic photo of PDLLA membrane. It showed the inner-structure of the PDLLA electrospun membrane and the diameter of the fiber was 10µm. The fiber surface is relatively smooth with a small pore structure. B. The release behavior of BMP-2. The release rate of the composite that was wrapped with the PDLLA electrospun membrane was lower too much than the hydrogel composites without the electrospun membrane. Figure 5 In vivo persistence and histology sections from rat subcutaneous tissue after implantation of hydrogel composites for 7, 14, 28, and 42 days. The system began to decrease gradually on the 14th days, and disappeared substantially on the 42th day. The obvious inflammatory cells could be seen on the 7th days after implantation and the inflammatory reaction gradually disappeared after 42 days. Figure 6 The photos of the surgical procedure. Decortication of the L4 - L5 spine or L5- L6 spine exposed with the electric scalpel was performed by high-speed bur and(a) the composites were planted into the areas (b was hydrogel composites group, c was hydrogel composite coated with electrospun nanofiber membrane composites) . Figure 7 Radiographs of defect site in the spine obtained by X-ray examination after surgery for 20 weeks. A certain amount of new bone was formed along the edge of the spine in all groups. The shadow area of new bone in group d was larger and the density was increased than other three groups. (group a was collagen/n-HA/ PECE hydrogel composite, group b was collagen/n-HA/PECE hydrogel composite coated with PDLLA electrospun membrane, group c was collagen/n-HA/BMP-2@PCEC/PECE hydrogel composite, and group d was collagen/n-HA/BMP-2@PCEC/PECE hydrogel composite coated with PDLLA electrospun membrane.) Figure 8 A. The cross sectio photos at 4 weeks, 12 weeks and 20 weeks after operation by Micro-CT; B. The sagittal section photos at 4 weeks, 12 weeks and 20 ACS Paragon Plus Environment

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weeks after operation by Micro-CT. Some new bone was generated along the edge of the polished parts. The mass about group d was more than the other groups. With the time increasing, the new bone mass was also increased and the structure of bone trabecula was generated. Simultaneously, the interface between the host bone and the new bone could not be distinguished and more regular trabecular bone morphology could be seen. But the interfaces could be distinguished clearly in others Figure 9 A. The local photomicrographs of histologic images about HE staining at 4 weeks, 12weeks and 20 weeks post-surgery; B. The local photomicrographs of histologic images about masson staining at 4 weeks, 12 weeks and 20 weeks post-surgery; C. The whole and local photomicrographs of histologic images at 20 weeks post-surgery. Abbreviations and signs used: Host bone(HB), new bone (NB), bone marrow (BM), collagen(Col).



Scheme1 Schematic illustrating about the thermo-sensitive hydrogel and PDLLA electrospun nanofiber membrane composites for the spinal fusion.


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Figure1 A. Structure and 1H-NMR spectrum of PCEC copolymer; B. The GPC spectrum of PCEC copolymer; C. Structure and 1H-NMR spectrum of PECE copolymer.



Figure2 A. The particle size distributions of prepared blank and BMP-2 loaded PCEC nanoparticles. The size of blank nanoparticles was 90 nm and the zeta potential was -29.13 mV. The size distribution turned a little wider after BMP-2 was loaded into the blank nanoparticles. The size and the zeta potential of nanoparticle loading BMP-2 did not change obviously compared with the blank PCEC nanoparticles; B. The TEM photo of the PCEC nanoparticles loading the BMP-2.


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Figure 3 A. The rheological characteristics of the hydrogel composite. The hydrogel composites turned to a gel state from a flow state while the G’ value was higher than G’’ at 33oC. The hydrogel composite had obvious thermo-sensitive; B. The morphologic photo of the hydrogel composite. The hydrogel composite owned the connected network porous structure.



Figure 4 A. The morphologic photo of PDLLA membrane. It showed the inner-structure of the PDLLA electrospun membrane and the diameter of the fiber was 10µm. The fiber surface is relatively smooth with a small pore structure. B. The release behavior of BMP-2. The release rate of the composite that was wrapped with the PDLLA electrospun membrane was lower too much than the hydrogel composites without the electrospun membrane. .


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Figure 5 In vivo persistence and histology sections from rat subcutaneous tissue after implantation of hydrogel composites for 7, 14, 28, and 42 days. The system began to decrease gradually on the 14th days, and disappeared substantially on the 42th day. The obvious inflammatory cells could be seen on the 7th days after implantation and the inflammatory reaction gradually disappeared after 42 days. (scale bar=100µm)



Figure 6 The photos of the surgical procedure. Decortication of the L4 - L5 spine or L5- L6 spine exposed with the electric scalpel was performed by high-speed bur and(a) the composites were planted into the areas (b was hydrogel composites group, c was hydrogel composite coated with electrospun nanofiber membrane composites)


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Figure 7 Radiographs of defect site in the spine obtained by X-ray examination after surgery for 20 weeks. A certain amount of new bone was formed along the edge of the spine in all groups. The shadow area of new bone in group d was larger and the density was increased than other three groups. (group a was collagen/n-HA/ PECE hydrogel composite, group b was collagen/n-HA/PECE hydrogel composite coated with PDLLA electrospun membrane, group c was collagen/n-HA/BMP-2@PCEC/PECE hydrogel composite, and group d was collagen/n-HA/BMP-2@PCEC/PECE hydrogel composite coated with PDLLA electrospun membrane.)



A

B

Figure 8 A. The cross sectio photos at 4 weeks, 12 weeks and 20 weeks after operation by Micro-CT; B. The sagittal section photos at 4 weeks, 12 weeks and 20 weeks after operation by Micro-CT. Some new bone was generated along the edge of the polished parts. The mass about group d was more than the other groups. With the time increasing, the new bone mass was also increased and the structure of bone trabecula was generated. Simultaneously, the interface between the host bone and the new bone could not be distinguished and more regular trabecular bone


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morphology could be seen. But the interfaces could be distinguished clearly in others.

Figure 9 A. The local photomicrographs of histologic images about HE staining at 4 weeks, 12weeks and 20 weeks post-surgery; B. The local photomicrographs of histologic images about masson staining at 4 weeks, 12 weeks and 20 weeks post-surgery; C. The whole and local photomicrographs of histologic images at 20 weeks post-surgery. Abbreviations and signs used: Host bone(HB), new bone (NB), bone marrow (BM), collagen(Col).



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Injectable and thermo-sensitive hydrogel and PDLLA electrospun

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