Microfiber-reinforced composite hydrogels loaded with rat adipose

Subscriber access provided by CAL STATE UNIV BAKERSFIELD

Tissue Engineering and Regenerative Medicine

Microfiber-reinforced composite hydrogels loaded with rat adipose-derived stem cells and BMP-2 for the treatment of medication-related osteonecrosis of the jaw in a rat model Haoran Ning, Xiaowei Wu, Qing Wu, Wanlu Yu, Huaiji Wang, Shang Zheng, Yunong Chen, Yongyong Li, and Jiansheng Su ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/ acsbiomaterials.8b01468 • Publication Date (Web): 12 Apr 2019 Downloaded from http://pubs.acs.org on April 16, 2019

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 36 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

Microfiber-reinforced composite hydrogels loaded with rat adiposederived stem cells and BMP-2 for the treatment of medication-related osteonecrosis of the jaw in a rat model Haoran Ning†, Xiaowei Wu†,‡, Qing Wu†, Wanlu Yu†, Huaiji Wang†, Shang Zheng†, Yunong Chen†, Yongyong Li*,§, Jiansheng Su*,†

† Department of Prosthodontics, School & Hospital of Stomatology, Tongji University, Shanghai Engineering Research Center of Tooth Restoration and Regeneration, Shanghai 200072, China. ‡ Department of Orthodontics, Peking University School and Hospital of Stomatology, Beijing 10081, China. § The Institute for Biomedical Engineering & Nano Science (iNANO), Tongji University School of Medicine, Shanghai 200092, China.

Corresponding Authors *Address: Department of Prosthodontics, School & Hospital of Stomatology, Tongji University, Shanghai Engineering Research Center of Tooth Restoration and Regeneration, Shanghai 200072, China. E-mail: [email protected] *Address: The Institute for Biomedical Engineering & Nano Science (iNANO), Tongji University School of Medicine, Shanghai 200092, China. Email: [email protected]

E-mail : [email protected] (Haoran Ning), [email protected] (Jiansheng Su)

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: Severe adverse reactions of bisphosphonates and antiresorptive or antiangiogenic medications, termed medication-related osteonecrosis of the jaw (MRONJ), have been reported. MRONJ are difficult to completely cure and could cause great pain to patients. Recent studies have shown that mesenchymal stem cell (MSC) therapies are effective for treating MRONJ, but the method of intravenous injection is unstable and increases the risk of producing tumors. In the present study, low-acyl gellan gum (LAGG) hydrogels were modified with hemicellulose polysaccharide microfibers (PMs) to improve the performance of supporting three-dimensional (3D) cell growth. LAGG-PM composite hydrogels were found to be nontoxic to rat adiposederived stem cells (rADSCs) in vitro. The hydrogels also promoted the secretion of angiogenic factors, induced osteoclastogenesis by conditioned medium and supported osteogenic marker expression after the addition of human bone morphogenetic protein2 (BMP-2). Due to its injectability, the LAGG-PM composite hydrogel incorporated with rADSCs and BMP-2 could be applied into the MRONJ lesion site, which promoted mucosal recovery, bone tissue reconstruction and osteoclastogenesis. This study confirms the potential applications of LAGG-PM composite hydrogels as 3D cell culture platforms and delivery vehicles for the treatment of MRONJ in a rat model. KEYWORDS: bisphosphonates, low-acyl gellan gum, three-dimentinal culture, osteogenesis, osteoclasts

2


Page 2 of 36

Page 3 of 36 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 Bisphosphonates(BPs) and some other antiresorptive or antiangiogenic medications, such as denosumab, sunitinib and bevacizumab, have been reported to cause jaw necrosis1-3, termed medication-related osteonecrosis of the jaw (MRONJ)4. The incidence of MRONJ in osteoporosis patients with oral BPs was 0.1-7%, while the incidence in cancer patients with intravenous BPs increased to 1.6-14.8%4. Therefore, populations with high risk for MRONJ are numerous and the potential harm should not be neglected. MRONJ often occurs after tooth extraction5. The clinical manifestations of MRONJ include maxillofacial pain, soft tissue swelling, bone exposure, inflammation caused by necrotic bone, tooth loosening and skin fistulas6. Surgical procedures have been shown to be difficult for defining the debridement boundary, which sometimes lead to even larger areas of exposure and pain of the infected bone7. The therapeutic efficiency of clinical treatment is limited, including surgical debridement, antimicrobial therapy, hyperbaric oxygen therapy, and immunosuppressive therapy8. Furthermore, due to the long half-life of BPs in bone (up to 11 years)9, short-term withdrawal cannot effectively eliminate the impact on the jaw. Considering the limitations of traditional treatments for MRONJ10, it is urgent to develop an effective treatment method to locally promote osteogenesis of the jaw or to prevent the occurrence of MRONJ. The pathogenesis of MRONJ has not been fully elucidated. Current hypotheses include inhibition of bone remodeling, angiogenesis inhibition, soft tissue injury, infection and immune factors4. Studies have reported that the accumulation of BPs inhibits the activity and function of oesteoclasts, osteoblasts and mesenchymal stem cells(MSCs) in vivo and in vitro, which suppresses bone formation and bone resorption11-16. The proliferation and angiogenesis of endothelial cell were also inhibited17. It was found that the level of angiogenic factors in metastatic breast cancer patients reduced by Zoledronic acid(Zol) significantly. Zol, a third-generation of nitrogen-containing BPs, were widely used in the treatment of osteoporosis and cancer patients18-19. Research has suggested that the proliferation, multidifferentiation capacities and osteoclastinducing ability of BMSCs derived from MRONJ lesion(central area of the osteonecrotic) severely decrease, while BMSCs derived from MRONJ lesion boundary(peripheral area of debridement boundary) are slightly inhibited15. Therefore, the suppression of MSCs is closely associated with MRONJ. MSCs possess the abilities of self-renewal, multidirectional differentiation20, and the secretion of growth factors and cytokines21-22, which provide a research direction for the treatment of MRONJ14. Investigators have demonstrated that intravenous or transplanted MSCs had certain therapeutic effects on MRONJ2324. The therapeutic effect of MSCs on MRONJ might depend on the ability of osteogenic differentiation14, stimulation of osteoclastogenesis and 14, 25 26 angiogenesis , and immune regulation . As progenitors of osteoblasts, MSCs were able to differentiate into osteoblasts and provide a supplementary source of osteoblasts for improving MRONJ27. The conditioned medium of MSCs was considered to be effective in repairing MRONJ, promoting osteogenic differentiation of MSCs and alleviating the inhibitory effect of Zol14. It also contributed to alleviating the suppression of ZoL on osteoclasts and regulating osteoclastogenesis25. On the other hand, it has been indicated that the therapeutic interactions between transplanted and diseased MSCs for healing MRONJ comes from mitochondria exchange28. 3



However, after intravenous injections, most of the administered MSCs were initially trapped in the lungs29, and only a small fraction of MSCs reached the bone marrow28. Furthermore, intravenous injection increased the risk of producing tumors30. Directly transplanted MSCs were plagued by low engraftment and poor survival31-32. Considering the general condition of patients, local treatment is a better choice. It is necessary to develop effective method to load MSCs to the lesion site, support its vitality and function, improve the microenvironment of jaw, and repair MRONJ. Hydrogels have shown applicability as an effective delivery vehicle of MSCs and growth factors for three-dimensional (3D) cell culture and tissue engineering33-34. Therefore, we developed a novel microfiber-reinforced gellan gum hydrogel loaded with rat adipose-derived mesenchymal stem cells (rADSCs) and human bone morphogenetic protein-2 (BMP-2) to treat MRONJ in a rat model. BMP-2 is not only an osteoinductive agent for regulating osteogenic differentiation of MSCs and bone regeneration35, but also a potent stimulator of new blood vessel formation36. It has been reported that that sustained controlled release of BMP-2 promotes the revascularization and early creeping substitution of osteonecrotic bone37. Therefore, BMP-2 was incorporated to hydrogel for promoting osteogenic differentiation of rADSCs, bone tissue regeneration and angiogenesis. In order to prevent the side effects of high concentration of BMP2, we have verified the optimal dose of BMP-2 in 3D culture. Intravenous injection of stromal vascular fraction cells isolated from adipose tissues has been proved to be a suitable treatment for MRONJ in mice38. Researchers have treated MRONJ with ADSCs24, which could be obtained from diverse sources and present proliferative capacity39, have been proved to promote angiogenesis and wound healing40. Studies have indicated that ADSCs are able to survive in ischemic environment, and release a large number of growth factors to promote angiogenesis41, which is consistent with osteonecrosis and ischemic injury environment of MRONJ. Gellan gum (GG) is a bacterial extracellular polysaccharide produced by Sphingomonas elodea. It is a negatively charged exopolysaccharide comprised of four repeating carbohydrates, including L-rhamnose, D-glucuronic acid and two D-glucose subunits42. GG was originally used as a food additive and was recently applied in tissue engineering due to its biocompatibility and low cytotoxicity43. At high temperatures, gellan molecules exist as random coils, and at low temperatures, they are present in double helices44. Helical sequences then aggregate to form cross-linking sites for the network, which is strongly stabilized by the multivalent cations. Divalent cations, such as Ca2+ and Mg2+, can form direct bridges between pairs of double helices43. Some scholars obtained selfsupporting GG hydrogel for encapsulation of rat bone marrow cells (rBMCs) by proportional addition of culture media into 1% GG solution45. However, we noted that rBMCs within the GG hydrogel could not form cell pseudopodia, and the long-term viability was not high. Therefore, we modified the low-acyl gellan gum (LAGG) hydrogel by adding hemicellulose polysaccharide microfibers (PMs) to enhance the mechanical strength of the LAGG hydrogel and to make the internal structure fibrous, which provided adhesion sites for rADSCs and promoted cell viability. Hemicellulose exists widely in plant cell walls. Both hemicellulose and LAGG are natural polymer with attractive features. They are renewable, nontoxic, biodegradable, and biocompatible44, 46. Xylan-type hemicellulose has been reported to have beneficial functions including inhibition 4


Page 4 of 36

Page 5 of 36 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


of cell mutation, anti-inflammatory, immune and anti-cancer47-50. We constructed a LAGG-PM 3D culture model in vitro to maintain the proliferation and secretion ability of rADSCs. Using this model, we observed the osteogenic differentiation capability of rADSCs by incorporating BMP-2 into the LAGGPM hydrogels. We further applied LAGG-PM hydrogels to test their efficacy in the treatment of rat MRONJ models. Furthermore, LAGG-PM hydrogels loading rADSCs with BMP-2 were transplanted into rat MRONJ models. The improved LAGG-PM hydrogels enabled the enhanced engraftment of rADSCs into the defect site and transport of the growth factor, significantly promoting mucosal healing, bone turnover and osteoclastogenesis at the lesion site. The LAGG-PM hydrogel constructs changed the microenvironment of bone tissue and improved tissue regeneration of MRONJ. Experimental Section Formation of LAGG-PM hydrogel Low-acyl gellan gum (LAGG-10G; Biomaterials USA LLC, Richmond, VA, USA) was dissolved in deionized water at 80 °C under constant agitation at a concentration of 1.1% (w/v). Hemicellulose polysaccharide microfibers (PMs; PFIBER5G, Biomaterials USA LLC, Richmond, VA, USA) were first dissolved in deionized water at room temperature and then mixed into LAGG hydrogels to reach a final concentration of 0.9%. LAGG and LAGG-PM hydrogels were obtained by adding alpha-MEM (αMEM, HyClone, USA) at a 1:1 volume ratio to LAGG or LAGG-PM solution followed by light agitation and maintained at 37 °C under 5% CO2 in a humidified incubator for 30 min. Characterization of LAGG and LAGG-PM hydrogels The gel states of LAGG and LAGG-PM before and after gelation were observed, and the gelation times were recorded. Fourier transform infrared (FTIR) spectra of the freeze-dried powders of LAGG and LAGG-PM hydrogels were then acquired using a Nicolet IS 10 spectrometer (Thermo Fisher, USA) in the wavenumber range between 4000 and 650 cm-1. To observe the internal morphology, the freeze-dried hydrogels were coated with gold and examined using a scanning electron microscope (SEM: S4800, Hitachi, Japan). To test the swelling and degradation properties, hydrogels were soaked in PBS at 37 °C. At predetermined timepoints (days 0, 1, 3, 7, 10, and 14), the wet weight (Ww) of samples was measured. Then, the samples were freeze-dried and weighed as the final dry weight (Wdf), and the day 0 samples were freeze-dried and weighed as the initial dry weight (Wdi). The swelling ratio (Ws) and weight loss (Wl) were calculated according to two equations as follows: Ws(%)=(Ww-Wdi)/Wdi×100 and Wl(%)=(Wdi-Wdf)/Wdi×10051. The elastic modulus was measured using an ARES-G2 rheometer (TA Instruments, USA) in a frequency scan mode of 0.110 Hz at a low strain (0.7%). rADSCs isolation and characterization Allogeneic rADSCs were isolated from subcutaneous adipose tissue in the inguinal groove of three 6-week-old female Sprague-Dawley (SD) rats24. Briefly, adipose tissues were harvested from the inguinal groove and cut into small fragments. Fat tissues were digested with 0.1% collagenase type I (Sigma, USA) in serum-free αMEM at 37 °C for 1 h. After the addition of an equal volume of 5



αMEM that contained 10% FBS, adipocytes were harvested by centrifugation. Then, the pellet was resuspended in αMEM containing 10% FBS, 50 U/mL penicillin and 50 mg/mL streptomycin and cultured at 37 °C under 5% CO2 in a humidified incubator. The culture medium was changed every 3 days, and the cells were used at passages 4-5. To characterize the rADSCs, the specific surface antigens were analyzed by flow cytometry as previously reported. Briefly, single-cell suspensions (1×106 cells) were incubated with 100 µl fluorescein isothiocyanate (FITC)-conjugated antibodies against rat CD34, CD45, CD29 and CD90 for 30 min at 4 °C. As a control, rADSCs were incubated without any target. Data acquisition and analysis were performed using a flow cytometer (BD Biosciences, San Jose, CA, USA) equipped with FlowJo software (TreeStar, OR). Cell-hydrogel 3D coculture LAGG and LAGG-PM hydrogel constructs were obtained by adding rADSCs (6×106 cells/ml) suspended in culture medium at a 1:1 volume ratio to LAGG or LAGG-PM solution followed by light agitation and maintained at 37 °C under 5% CO2 in a humidified incubator for 30 min. After gelation, the hydrogel constructs were cultured in αMEM with 10% FBS, 50 U/mL penicillin and 50 mg/mL streptomycin for 1, 3, 7, and 14 days. The spreading and morphology of rADSCs were observed under an optical microscope (Nikon eclipse 80i, Japan). The viability of rADSCs loaded inside the LAGG and LAGG-PM hydrogels was evaluated using a live/dead viability/cytotoxicity kit (Molecular Probes, ThermoFisher, USA) following the protocol provided by the manufacturer. Live (green) cells were labeled with calcein AM, and dead (red) cells were labeled with ethidium homodimer-1. The stained cells were imaged by Carl Zeiss Microscopy (USA), and the quantification of cell viability was calculated by manually counting the images (n=4). The viability and proliferation of hydrogel constructs were evaluated by a cell counting kit-8 (CCK-8) assay. The LAGG or LAGG-PM solution was mixed with the cell suspension (final concentration of 3×106 cells/ml) at a 1:1 volume ratio, and the pre-gelation solution was then added into 96-well plates (50 μL per well) and maintained at 37 °C under 5% CO2 in a humidified incubator for 30 min. After 1, 3, 7, and 14 days, the culture medium was removed, and the hydrogel constructs were treated with 10% CCK-8 reagent (Beyotime, Shanghai, China) in culture medium (100 µL per well) for 2 h in the dark at 37 °C. The mixture was transferred to a new 96-well plate, and the absorbance was measured at a wavelength of 450 nm using a microplate reader52. The levels of vascular endothelial growth factor (VEGF), recombinant human insulin-like growth factor I (IGF-I) and nuclear factor κ-B ligand (RANKL) in conditioned medium were measured with ELISA. To collect conditioned medium, the pre-gelation solution of LAGG or LAGG-PM was added into 24well plates (200 μL per well). After 3, 7, and 10 days, the culture media of hydrogel constructs was collected from the 24-well plates and stored at -80 °C, and fresh media was then added. The concentrations of VEGF, IGF-I and RANKL were measured with rat VEGF ELISA kits, rat IGF-I ELISA kits and rat RANKL ELISA kits (Boster Biological Technology, Wuhan, China). The conditioned medium of hydrogel constructs at 7 days and 20 ng/ml RANKL were added to 24-well plates to treat RAW264.7 cells. After 7 days of treatment, the osteoclastogenesis of RAW264.7 cells was detected by tartrate-resistant acid phosphatase (TRAP) staining using an acid phosphatase leukocyte kit (Sigma6


Page 6 of 36

Page 7 of 36 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


Aldrich, USA). The number of osteoclasts was calculated under a high-power field (X200). rADSCs differentiation within LAGG-PM hydrogel To test the optimum concentration of bone morphogenetic protein-2 (BMP-2) for the osteogenic differentiation capability of rADSCs inside LAGG-PM hydrogels, recombinant human BMP-2 (Novus, USA) was added to the pre-gelation solution at different final concentrations of 0, 25, 50, 100 or 200 ng/ml. After culturing rADSCs within LAGG-PM hydrogels in 24-well plates for 7 days, the hydrogel constructs were washed with PBS three times and homogenized with a pestle in TRIzol reagent (TAKARA BIO INC., Japan). Total RNA was then extracted according to the manufacturer’s instructions and quantified with a microspectrophotometer (NanoVue Plus, Biochrom Ltd, UK). RNA was reversetranscribed into single-stranded cDNA using a PrimeScript RT Master Mix (RR036A, TAKARA BIO INC, Japan). Subsequently, real-time quantitative polymerase chain reaction (qRT-PCR) was performed on a LightCycler 96 (Roche Diagnostic, USA) using FastStart Universal SYBR Green Master Mix (Roche Diagnostics, USA) according to the manufacturer’s recommendations for the receptor activator of collagen type I alpha 1 (COL Ia1), alkaline phosphatase (ALP) and glyceraldehyde-3-phosphatedehydrogenase (GAPDH) using the comparative cycle threshold (CT) method. All experiments were performed in triplicate. To examine the osteogenic differentiation of rADSCs, hydrogel constructs loaded with BMP-2 (50 ng/ml) were cultured for 3, 7, and 10 days. As previously reported, qRT-PCR was performed on markers of COL Ia1, ALP, VEGF, runtrelated transcription factor 2 (Runx2), osteocalcin (OCN), RANKL, osteoprotegerin (OPG) and GAPDH, which are listed in Table 1. In vitro BMP-2 release from LAGG and LAGG-PM hydrogels To examine the release of BMP-2 from LAGG and LAGG-PM hydrogels, BMP2 was loaded within hydrogels at a final concentration of 50 ng/ml. The hydrogels loaded with BMP-2 were incubated in PBS for 72 h at 37 °C. At each timepoint, the PBS release medium was removed and replaced with fresh PBS. The amount of BMP-2 in the release medium at each timepoint was determined using a human BMP-2 ELISA Kit (Boster Biological Technology, Wuhan, China). Transplantation of rADSCs using LAGG-PM hydrogel in a rat MRONJlike model This study was performed in accordance with the guidelines and regulations for the care and use of laboratory animals of the National Institutes of Health. All surgical procedures were approved by the Institutional Animal Care and Use Committee of Tongji University (Shanghai, China). MRONJ was induced in a rat model as previously described. Sixty-five (65) SD rats (8-week-old females) were administered Zol of 80 μg/kg/week intravenously via the tail vein for 2 weeks in the BP-treated group. Thereafter, the maxillary right first and second molars were extracted using a sharp dental explorer under deep anesthesia induced by intraperitoneal injection of ketamine hydrochloride (100 mg/kg) and xylazine (5 mg/kg). Subsequently, drugs were continuously administered intravenously for an additional 8 weeks after the extraction of the teeth. We defined only the rats that experienced bone exposure as the MRONJ model. Eight weeks after extraction, the rats with bone exposure were randomly divided into five groups as follows: (1) Control group (each extraction socket was drilled 7



using a 2.0 mm round bur to eliminate the necrotic bone and produce a 2×2×2 mm3 socket); (2) rADSCs I.V. group (intravenous administration of 4.8×104 rADSCs via the rat tail vein); (3) LAGG-PM group (8 µl LAGG-PM hydrogel was transplanted into each socket); (4) LAGG-PM+rADSCs group (8 µl LAGGPM hydrogel loaded with 2.4×104 rADSCs was transplanted into each socket); and (5) LAGG-PM+rADSCs+BMP2 group (8 µl LAGG-PM hydrogel loaded with 2.4×104 rADSCs and 0.4 ng BMP-2 was transplanted into each socket). After surgical debridement in each group, transplantation was performed and the wound was sutured. A week later, the suture was removed. Half of the rats were euthanized 4 weeks following surgery, and the other half of rats were euthanized 8 weeks after surgery. Micro-CT analysis The maxillary bones were dissected, fixed in 4% paraformaldehyde (PFA), and scanned with 10 μm isotropic voxels on a microcomputed tomography (microCT) system (Scanco Medical, Zurich, Switzerland). Using analysis software (Start Xming), three-dimensional images were reconstructed, and bone morphometric analyses of the extraction sockets were performed from various sections. Bone morphometric indices of bone volume fraction(BV/TV) and bone mineral density(BMD) were measured. Histological analysis The specimens were decalcified with 10% EDTA solution for 3 months and dehydrated with graded concentrations of ethanol, processed in xylene, and then embedded in paraffin. The samples were cut buccolingually at the tooth extraction area and then stained with hematoxylin-eosin (HE) and Masson’s trichrome (MT). Osteoclasts were identified by TRAP staining using an acid phosphatase leukocyte kit (Sigma-Aldrich, USA). The histology was observed and analyzed using optical microscopy (Nikon eclipse 80i, Japan). The percentages of empty osteocyte lacunae and the average number of osteoclasts were counted under a high-power field (X200). Sequential fluorescence image analysis To label calcium deposition in extracted teeth, tetracycline hydrochloride, calcein and alizarin red were administered intravenously at 2, 4, and 7 weeks after surgery. All rats were euthanized at 8 weeks, and the samples were dehydrated in graded concentrations of ethanol from 50% to 100% and embedded in poly (methyl methacrylate) without decalcification. The specimens were cut and ground to a thickness of 35 μm, polished to a thickness of 20 μm and then observed under a laser scanning confocal microscope (LSCM, Nikon, Japan). The area of fluorochrome-stained bone was quantified with ImageJ software (NIH, Bethesda, MD, USA). Statistical analysis Data were expressed as the mean ± standard deviation. All experiments were performed independently in triplicate. Statistical analysis was performed using unpaired Student’s t-tests and Tukey’s test. P values < 0.01 and P values < 0.05 were considered statistically significant. Results and discussion Physical and mechanical characterization of LAGG and LAGG-PM hydrogels 8


Page 8 of 36

Page 9 of 36 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


LAGG and LAGG-PM solutions and αMEM were mixed in equal volume to form the hydrogels. LAGG molecules automatically aggregated from random coiled structures to paired helical structures when the LAGG solution was cooled43. Cations within αMEM formed direct bridges between pairs of double helices, of which the concentrations were as follows: CaCl2 (1.8 mM), KCl (5.4 mM), MgSO4 (0.81 mM), NaCl (116 mM), and NaH2PO4 (1 mM)45. It has been reported that the cations in αMEM were sufficient to cause gelation of 1% GG solution45. Polysaccharide microfibers were used as a reinforcement phase for composite hydrogel formation. Due to the carboxyl groups on the fibers, PMs were linked to the cations in αMEM and LAGG molecules, which increased the rate of the gelation process. The state of hydrogels before and after gelation was shown in Figure 1A. After gelation, no liquid flow was observed, and hydrogels turned red with the addition of the culture medium. The loss of fluidity after gelation indicated that the gel had formed. The addition of PMs decreased the gelation time of the LAGG hydrogel (Figure 1B). LAGG and LAGG-PM powders were characterized with FTIR (Figure 1C). As illustrated in the FTIR absorption spectrum, the characteristic peaks at approximately 3288 cm-1 (O–H stretching), 1615 cm-1 (asymmetric COO– stretching), 2923 cm-1 and 1405 cm-1 (-CH- stretching), and 1024 cm-1 (C–O stretching) were observed in both LAGG and LAGG-PM. The FTIR spectra of LAGG-PM revealed the appearance of new peaks at 890 cm-1 was attributed to β-glycosidic bond.The results indicated the successful incorporation and distribution of PMs into the LAGG matrix. SEM images of freeze-dried samples revealed connected pore structures within LAGG and LAGG-PM hydrogels (Figure 1D). SEM showed that the lyophilized LAGG hydrogels had a typical spongy interior morphology with open macropores and anisotropic porosity. The LAGG-PM hydrogels had relatively uniform pore structures, and many microfilaments were dispersed in the wall structure, which provided adhesion sites for cells. The pore size of the hydrogels was calculated from the SEM images, which revealed no statistically significant difference in the pore sizes between the two types of hydrogels (Figure 1E). Figure 1F and 1G shows the swelling properties and degradation of LAGG and LAGG-PM hydrogels incubated in PBS at 37 °C. The results revealed that the value of water uptake in LAGG hydrogels was greater than that in LAGG-PM hydrogels. Because the increased crosslink density was consistent with a tighter matrix, the LAGG-PM hydrogels were less prone to swelling. Regarding the weight loss of the hydrogels, the degradation of LAGG hydrogels was much more rapid than that of LAGG-PM hydrogels. After 14 days, the weights of LAGG hydrogels decreased to 32.9±4.9% of initial mass, while LAGG-PM hydrogel degraded to 63.4±4.5%. This was due to the dense microfiber structures and the low hydration and slow degradation rate of hemicellulose polysaccharide microfibers. Due to the high water content or very low polysaccharide content, the amount of enzymes needed to degrade the hydrogel is quite low. The glycoside bond of polysaccharide hydrolyzed in PBS, so the degradation speed was relatively faster than high strength hydrogel. Figure 1H and 1I shows the rheological properties of LAGG and LAGG-PM hydrogels. The storage moduli (G′) and loss moduli (G″) of the hydrogels were measured across a frequency range (0.1-10 Hz). The G′ of LAGG and LAGG-PM hydrogels showed a slight linear increase and were constantly higher than G″. The results indicated that the tested hydrogels had a certain viscosity and elasticity. The average storage modulus at 1 Hz of LAGGPM hydrogels (123±5.1 Pa) was much greater than that of LAGG hydrogels 9



(23.2±2.4 Pa), which could be attributed to the greater degree of crosslinking of LAGG-PM hydrogels and dense microfibers inside the LAGG-PM hydrogels. In our study, the low-strength hydrogels were not used as load-bearing scaffolds, but were mainly used as a delivery vehicle to load rADSCs and support the activity of rADSCs, thus promoting local formation of normal bone microenvironment. Biological behavior of rADSCs within LAGG and LAGG-PM hydrogels Adherent rADSCs appeared in the cell culture dishes after culturing for 48 h. After three passages, rADSCs became spindle-shaped and developed into many clusters. Flow cytometry analysis showed that rADSCs at passage 3 were positive for the MSC surface markers CD29 and CD90 and negative for the hematopoietic markers CD34 and CD45 (Figure 2A). Biocompatibility is critical for biomaterials used in 3D cell and tissue culture. To evaluate the cell morphology and viability of rADSCs inside LAGG or LAGG-PM hydrogels, cell-hydrogel constructs were cultured for 1, 3, 7, and 14 days. The optical images showed that at 1 and 14 days after coculture of cells inside the hydrogels, rADSCs appeared round within LAGG hydrogels at all of the observed timepoints. In contrast, in LAGG-PM hydrogels, cellular processes extended and spread extensively up to 14 days, which was also confirmed by live/dead staining (Figure 2B). Confocal images showed that rADSCs were highly viable and distributed homogenously during the 3D cell culture period (Figure 2C). Visual examination showed that the live cell density of different types of hydrogel constructs was similar. The results demonstrated that the cell viability was maintained within the hydrogels over a 14-day culture period and was maintained well when PMs were incorporated. PMs improved the permissiveness of hydrogels and provided more cell binding sites. The proliferation of rADSCs inside LAGG or LAGG-PM hydrogels was evaluated by a CCK-8 assay at different timepoints over a period of 2 weeks(Figure 2D). As shown in Figure 2D, the OD values decreased from 1 to 3 days and gradually increased from 3 to 14 days. The cells in each group exhibited similar growth characteristics. However, the OD value of the LAGG-PM group was significantly greater than that of the LAGG group. The decrease in the OD values from 1 to 3 days might be due to the loss of unattached rADSCs after changing the culture medium. Figure 2C showed that the number of dead cells did not increase significantly at 3 days, which supported this hypothesis. However, there was no significant decrease in the number of living cells, probably due to the uniform distribution of rADSCs within the hydrogel, and fluorescence images showed the effect of multilayer cell stacking. The percentage of live cell was shown in Figure 2E, which revealed both groups maintained a high percentage of living cells. The results revealed that PMs promoted the proliferation and adhesion of rADSCs. Compared with LAGG hydrogels, microfiber-reinforced LAGG-PM hydrogels improved biocompatibility and provided a more favorable microenvironment for rADSCs. The preparation of LAGG-PM hydrogel constructs was convenient for application, and the 3D culture system could be constructed in vitro to support cell physiological maintenance. To test the secretion function of rADSCs during the 3D culture period, the secretion of VEGF, IGF-I and RANKL in conditioned medium was investigated by ELISA at 3, 7 and 10 days (Figure 2F-H). Figure 2F showed that VEGF secretion in the LAGG group gradually decreased with time, while VEGF secretion in the LAGG-PM group increased for 7 days and then decreased. At 7 10


Page 10 of 36

Page 11 of 36 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


and 10 days, secreted VEGF in the LAGG-PM group was significantly greater than that in the LAGG group. The level of VEGF secretion in LAGG-PM group was 1598±17.2~2126±118.8 pg/ml, which was slightly higher than other 3D culture study53. Figure 2G revealed that IGF-1 in the LAGG group increased with time, and the LAGG-PM group increased first and then decreased, but each assayed timepoint for the LAGG-PM group was significantly greater than that for the LAGG group. The secretion level of IGF-1 in LAGG-PM group was 1746±255.9~5955.8±372.9 pg/ml, which was significantly higher than IGF-1 level (1515.60+211.83) secreted by hBMSCs after 48 h. The trend of secreted VEGF and IGF-1 protein levels were similar to previous studies. Kim et al.54 observed that the secretion of VEGF from genetically engineered MSCs increased from 2 to 8 days but decreased from 8 to 14 days. Kaibuchi et al.55 observed that the levels of secreted VEGF in bone marrow-derived MSCs gradually decreased from 2 to 7 days. Birnbaum et al.56 found that IGF-1 mRNA expression levels in osteoblasts increased from 5 to 11 days and declined thereafter. Park et al.53 utilized a catechol-functionalized hyaluronic acid hydrogel to enhance stem cell-mediated angiogenesis, but the level of VEGF secreted from hADSCs decreased over time. VEGF and IGF-1 stimulated angiogenesis by stabilizing newly formed blood vessels and endothelial cell tubes57. In addition, IGF-1 regulated the balance of osteoblasts and osteoclasts, which was conducive to bone remodeling58. Studies have reported that conditioned medium could promote angiogenesis, epithelial healing and bone turnover59-60. Ogata et al.14 treated rat MRONJ by intravenous injection of conditioned medium of MSCs. It was found that growth factors, cytokines and paracrine factors secreted by MSCs had therapeutic effects on MRONJ14. The effect of cell-hydrogel constructs for inducing osteoclastogenesis was assessed. Figure 2H showed the secretion of RANKL in two groups increased with time, and the LAGG-PM group was significantly greater than that in the LAGG group. The level of RANKL secretion in LAGG-PM group was 15.8±0.6~27.8±0.7 pg/ml, which was similar to the clinical level. Wu et al.61 measured the level of RANKL in serum of patients after orthognathic surgery by ELISA, which was about 40-120 pg/ml. Figure 2I-J revealed that TRAP-positive multinucleated giant cells were observed in LAGG-PM group, and the number of which increased significantly. Conditioned medium promoted osteoclastic differentiation of RAW264.7 cells, which might be attributed to cytokines in conditioned medium. Ogata et al.25 indicated that conditioned medium of MSCs regulated osteocalast differentiation, and reduced the effect of RANKL inhibitor. The LAGG-PM-rADSCs constructs promoted the secretion of angiogenic factors and induced osteoclastogenesis by conditioned medium. The results suggested that rADSCs in LAGG-PM hydrogels maintained the capability of the paracrine secretion of cytokines, which has been reported to be important for bone turnover21. Differentiation of rADSCs within LAGG-PM hydrogel and BMP-2 release in vitro In addition to supporting the growth of rADSCs, hydrogels can also be a delivery vehicle for growth factors. To enhance the intrinsic osteoinductivity of LAGGPM hydrogels, the osteogenic growth factor BMP-2 was incorporated. Electrostatic interactions formed between the BMP-2 and the LAGG molecules. BMP-2 is an important positive regulator of MSC osteogenic differentiation induction, and many researchers have tethered it to scaffolds to stimulate osteogenesis35. Because the differentiation of MSCs in 2D and 3D culture is 11



different62, we tested the effect of different BMP-2 concentrations on rADSC differentiation within LAGG-PM hydrogels. qRT-PCR experiments were performed to test the optimum concentration of BMP-2 after 7 days. The results showed that the expression of COL Ia1 and ALP was upregulated with increasing BMP-2 concentrations, up to 50 ng/ml, and then downregulated as the concentration of BMP-2 increased to 200 ng/ml (Figure 3 A, B). The expression levels of COL Ia1 and ALP at 50 ng/ml were significantly greater than those in the other groups, which revealed the optimum concentration of BMP-2. The optimal concentration of BMP-2 was less than that reported in some studies63-64, but it was consistent with other reports37, 65. The optimum concentration was a low dose, possibly because LAGG-PM hydrogels prolonged the retention of BMP-2, and rADSCs responded differently in 3D conditions. Considering the side effects of high doses of BMP-2, researchers have also chosen low doses (20 ng/ml) as the therapeutic constant release concentration37. We tested the release of BMP-2 from LAGG and LAGG-PM hydrogels using a human BMP-2 ELISA Kit. Approximately 82±2.6% of the initially loaded BMP-2 was released from the LAGG hydrogel at 24 h, and the burst release of the LAGG-PM hydrogel was significantly reduced to 58.7±0.8%. After 72 h, almost 91.3±2.1% BMP-2 was released in the LAGG hydrogel, while approximately 30% BMP-2 was retained in the LAGG-PM hydrogel (Figure 3 C). Hydrogels had a high burst release and sufficient release of BMP-2, possibly because BMP-2 was physically loaded into the hydrogel. The release rate was affected by the degradation rate of hydrogels; therefore, the BMP-2 release rate from the LAGG-PM hydrogel was slower than that from the LAGG hydrogel. Kempem et al.66 reported that the biological activity of BMP-2 released by composite materials was higher than that of BMP-2 added directly to the control cultrures at corresponding concentration. Further, qRT-PCR was performed to examine the osteogenic and osteoclastic gene expression of rADSCs inside the 3D LAGG-PM hydrogel loaded with BMP-2 (50 ng/ml) after 3, 7 and 10 days. The early-stage osteoblastic genes, including COL Ia1, ALP and Runx2, increased over time and were upregulated in the LAGG-PM-BMP2 group. The expression levels of COL Ia1 and ALP in the LAGG-PM-BMP2 group were significantly greater than those in the LAGGPM group. Runx2 only exhibited significantly greater expression than that in the LAGG-PM group at 7 and 10 days. To investigate the change in the late-stage osteogenic gene expression, OCN was upregulated significantly in the LAGGPM-BMP2 group at 10 days (Figure 3D-G). Therefore, the addition of BMP-2 promoted the osteogenic differentiation of rADSCs. The expression of angiogenesis gene VEGF was up-regulated in LAGG-PM-BMP2 group, but decreased with time(Figure 3H). BMP-2 is not only an osteoinductive agent for bone regeneration35, but also a potent stimulator of new blood vessel formation36. Notably, the expression of RANKL and OPG increased progressively with time in both groups, which was significantly enhanced by BMP-2. In addition, the ratio of RANKL/OPG expression decreased significantly at 3 days and significantly increased at 10 days (Figure 3I-K). It has been reported that BMP2 increases RANKL and OPG expression and the RANKL/OPG expression ration in chondrocytes, thus promoting osteoclastogenic activity67. In our study, effects of combinations of BMP-2 and LAGG-PM hydrogel have been verified, which could induce osteogenic and angiogenic gene expression of rADSCs, and promote the expression of RANKL. It was speculated that the LAGG-PM 12


Page 12 of 36

Page 13 of 36 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


hydrogel loaded with BMP-2 had bone bioactivity, which could regulate the microenvironment of bone tissue and accelerate bone turnover. The establishment of a MRONJ-like model in SD rats As previously reported, we established a model of MRONJ-like disease in SD rats after the intravenous injection of Zol and tooth extraction. Zol is the third generation of BPs and is widely used in clinical practice as well as in the establishment of MRONJ animal models14, 55. MRONJ was definited as exposed bone in the maxillofacial region over 8 w in patients with treatment of antiresorptive or antiangiogenic medications4. Figure 4A shows a schematic diagram of the experimental modeling protocol. The first and second maxillary molars were removed 2 weeks after Zol injection (80 μg/kg/week) via the tail vein, and the model was completed after 8 weeks of continuous Zol administration. Eight weeks after tooth extraction, the macroscopic observation of the specimens revealed that 61.5% (40/65) of rats had exposed bones in the BP-treated group; in contrast, all of the mucous membranes healed in the untreated group, and the bone exposure rate was 0% (0/4) (Figure 4B, C). In terms of cross-sections, micro-CT analysis showed that the bone tissues in extraction sockets of the BP-treated group were unhealed, while the extraction sockets of the untreated group presented high-density shadows, and the bone tissue healed completely (Figure 4D). We circled a cylinder through the white circle along the alveolar socket as Volume of Interest (VOI) for measuring bone morphometric indices (Figure 4E). Figure 4F showed BV/TV in the BP-treated group decreased significantly, which due to the unrepaired bone tissue in alveolar fossa. Figure 4G suggested that the bone mineral density of BP-treated group was significantly increased under the injetion of ZOL, as demonstrated by Masson staining.Histological images revealed that the BP-treated group had open sockets with unhealed mucosa, and the connective tissue collapsed. There were large amounts of necrotic bones, empty bone lacunae, inflammatory cell infiltration and few osteoclasts. In the untreated group, mucosa healed completely with intact epithelial coverage and connective tissue and new bone formation in the extraction sockets. Masson staining showed that the collagen fibers in BP-treated group were few and disordered. The surrounding bone tissues were stained red, suggesting that bone tissues were mature and sclerotic.The number of osteoclasts increased, which indicated normal bone regeneration (Figure 4E-H). Figure 4I shows that the percentage of empty osteocyte lacunae counted in the BP-treated group significantly increased from 6.2 ± 2.5% to 74.4 ± 5.1%. Figure 4J shows the count of the number of osteoclasts per high-power field (X200) of TRAP staining, and the number of osteoclasts in the BP-treated group was significantly reduced. Based on these experimental results, we successfully established a rat model of MRONJ similar to human MRONJ disease by tail vein injection of Zol and tooth extraction, which included unhealed mucosa and necrotic bone exposure. Transplantation of rADSCs using LAGG-PM hydrogel promoted mucosal and bone regeneration in a rat model of MRONJ To confirm the regenerative potential of LAGG-PM hydrogels loaded with rADSCs, the therapeutic efficacy was evaluated in a rat MRONJ model. We only defined the rats that caused bone exposure as the MRONJ model and treated them accordingly. Four weeks after transplantation, the mucosa of rats was observed, and half of the samples were collected randomly. As shown by the macroscopic 13



observations, the LAGG-PM-rADSCs-BMP2 group demonstrated complete healing of the mucosa with a soft tissue covering and a bone exposure rate of 0% (0/9), while most of the Control group revealed unhealed exposed bone and mucosa, with a bone exposure rate of 66.7% (4/6). In the rADSCs I.V. group, LAGG-PM group and LAGG-PM-rADSCs group, the samples were partially healed, and others had visible necrotic bone. The bone exposure rates were 33.3% (2/6), 12.5% (1/8) and 12.5% (1/8), respectively (Figure 5A, B). Micro-CT revealed the regeneration of bone defects. The results indicated that osteogenesis occurred in the LAGG-PM-rADSCs group and the LAGG-PM-rADSCs-BMP2 group at the extraction sockets (Figure 5C). Quantitative analysis of BV/TV confirmed that the volume of new bone tissue increased significantly in LAGGPM-rADSCs-BMP2 group(Figure 5D). Compared with Conrol group, LAGGPM-rADSCs-BMP2 group showed a significant decrease in bone mineral density. Masson staining showed that in Control group the bone tissue around the extraction socket stained red, and there were a few in LAGG-PM group, while the other groups were normal, suggesting the effect of rADSCs on regulating the microenvironment of bone tissue. Histological analysis showed that the Control group had open sockets with the inward collapse of the epithelium and connective tissue. Large numbers of necrotic bones and empty osteocyte lacunae were found at the extraction sockets, while osteoclasts were rarely observed. In the rADSCs I.V. group and LAGG-PM group, the epithelium and connective tissue were partially repaired but not fully healed. Large numbers of inflammatory cells, necrotic bones and empty osteocyte lacunae were observed, while osteoclasts were still rarely observed. The LAGG-PM hydrogel wrapped the necrotic bone. In the LAGG-PM-rADSCs group and LAGG-PM-rADSCsBMP2 group, the mucosa completely healed to form soft tissue closures, and the residual LAGG-PM hydrogels were visible at the extraction site. Local bone formation was observed, and the number of osteoclasts significantly increased. More bone matrix deposits were observed at the bottom of the extraction site of the LAGG-PM-rADSCs-BMP2 group (Figure 5F-I). Figure 5K showed that the percentage of empty osteocyte lacunae counted in the LAGG-PM-rADSCs group and LAGG-PM-rADSCs-BMP2 group significantly decreased. Figure 5J, L revealed a count of the number of osteoclasts per high-power field (X200) of TRAP staining, and the number of osteoclasts in the LAGG-PM-rADSCs group and LAGG-PM-rADSCs-BMP2 group significantly increased. The experimental results showed that the transplantation of rADSCs and BMP-2 using the LAGG-PM hydrogel promoted mucosal healing, bone regeneration and osteoclastogenesis in the rat model of MRONJ. BPs have been reported to inhibit the osteogenic and angiogenic gene expression of rMSCs, including RUNX2, OCN and VEGF, while promoting OPG and inhibiting RANKL expression to inhibit the formation and activity of osteoclasts14, 68. In our study, LAGG-PM hydrogels provided microenvironments for rADSCs to promote paracrine factor secretion. It has been reported that paracrine factors could influence host cells to accelerate wound closure during wound healing and improve new blood vessel formation69. In addition, LAGG-PM hydrogels also contributed to the healing of mucosa and re-epithelialization. Studies have reported that hemicellulose have anti-phlogistic effects50. Cerqueira et al.69 assembled gellan gum-hyaluronic acid hydrogels, hADSCs and microvascular endothelial cells for promoting skin wound closure and vascularization. VEGF and IGF-1 stimulated angiogenesis by stabilizing newly formed blood vessels 14


Page 14 of 36

Page 15 of 36 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


and endothelial cell tubes57. Conditioned medium of LAGG-PM-rADSCs constructs promoted osteoclastic differentiation of RAW264.7 cells, which might be attributed to cytokines in conditioned medium, such as IGF-1 and RANKL. LAGG-PM hydrogels enhanced stem cell-mediated angiogenesis and osteoclastogenesis, which contributed to mucosal healing and bone regeneration in MRONJ. Furthermore, LAGG-PM hydrogels, which served as a delivery vehicle of BMP-2, enhanced the intrinsic osteoinductivity, which supported the osteogenic and angiogenic differentiation of rADSCs. As previously reported, BMP-2 within MSC-laden gelatin-methacryloyl hydrogels with 3D printed microchannels were found to promote osteoclast invasion, vascularization and heterotopic bone formation following implantation70. Wang et al.37 demonstrated that the controlled release of BMP-2 could induce callus wrapping of necrotic bone in the early stage and promote angiogenesis and substitution within the necrotic bone. Previous reports have indicated that the intravenous administration of MSCs has therapeutic effects on mouse and pig models of MRONJ23, 71. Due to different experimental conditions, tail vein injection of MSCs was still studied as a control group55. Our results showed that the intravenous administration of rADSCs was less effective, mainly because the number of rADSCs transplanted was extremely lower than that reported in other studies, which demonstrated the vital role of the LAGG-PM hydrogels. Because of superior tissue adhesiveness, the LAGG-PM hydrogel mediated the efficient engraftment of rADSCs and BMP-2 into the MRONJ defect. The transplantation significantly promoted mucosal healing, bone reconstruction and osteoclastogenesis. Sequential fluorescence image analysis of LAGG-PM hydrogel loaded with rADSCs in a rat model of MRONJ after 8 weeks. Figure 6A shows the new bone formation and mineralization area of tetracycline hydrochloride, calcein, and alizarin red markers at 2, 4, and 7 weeks using a sequential fluorescent labeling technique. To observe more information on the two extraction sockets, the sample was cut in the mesial-distal direction. The large image was scanned and spliced by 24 low-magnification images (X40). The newly formed bone was quantified by calculating the percentage of the area of fluorochrome-stained bone (Figure 6B-D). The yellow tetracycline hydrochloride, green calcein, and red alizarin red labels were statistically greater in the four experimental groups than those in the Control group. The LAGG-PMrADSCs-BMP2 group showed a significant increase in bone tissue production at each timepoint over 8 weeks. Conclusions In this study, we developed polysaccharide-reinforced hydrogels with promising potential for the treatment of a rat model of MRONJ. Our results revealed that the appropriate incorporation of PMs into LAGG could promote crosslinking degrees of hydrogels and mechanical strength. The spreading, viability and proliferation of rADSCs within 3D LAGG-PM hydrogels were enhanced. Due to the secretion of VEGF and IGF-1, GG-PM hydrogels enhanced rADSC-mediated angiogenesis. The cytokines in conditioned medium promoted the differentiation of osteoclast progenitor cells into osteoclasts, thus accelerating bone turnover. Furthermore, the LAGG-PM hydrogels supported the osteogenic differentiation 15



of rADSCs after the addition of BMP-2 (50 ng/ml). In vivo assays further validated that the transplantation of rADSCs using LAGG-PM hydrogel promoted mucosal healing, bone regeneration and osteoclastogenesis in a rat model of MRONJ. Our study suggested that the LAGG-PM hydrogels could offer a favorable platform for the application of in vitro three-dimensional cell culture and in vivo MRONJ-like disease treatment. Author information Corresponding Authors *Address: Department of Prosthodontics, School & Hospital of Stomatology, Tongji University, Shanghai Engineering Research Center of Tooth Restoration and Regeneration, Shanghai 200072, China. E-mail: [email protected] *Address: The Institute for Biomedical Engineering & Nano Science (iNANO), Tongji University School of Medicine, Shanghai 200092, China. Email: [email protected] ORCID Haoran Ning:0000-0002-8281-9102 Notes There are no conflicts to declare. Acknowledgements This work was supported by National Natural Science Foundation of China (No. 81572114, 81873715), Science and Technology Commission of Shanghai Municipality (No. 17140903600, 18441902100). We thank professor Xuejun Wen from the Institute for Biomedical Engineering & Nano Science (iNANO), Tongji University for his generous help with the design and synthesis of hydrogels.

Notes and references 1. Diniz-Freitas, M.; Fernández-Feijoo, J.; Diz, P.; Pousa, X.; Limeres, J., Denosumab‐related osteonecrosis of the jaw following non‐surgical periodontal therapy: A case report. J. Clin. Periodontol. 2018, 45 (5), 570-577. https://doi.org/10.1111/jcpe.12882 2. Brunello, A.; Saia, G.; Bedogni, A.; Scaglione, D.; Basso, U., Worsening of osteonecrosis of the jaw during treatment with sunitinib in a patient with metastatic renal cell carcinoma. Bone 2009, 44 (1), 173-175. https://doi.org/10.1016/j.bone.2008.08.132 3. Erovigni, F.; Gambino, A.; Cabras, M.; Fasciolo, A.; Fusco, V., Delayed Diagnosis of Osteonecrosis of the Jaw (ONJ) Associated with Bevacizumab Therapy in Colorectal Cancer Patients: Report of Two Cases. Dent. J. 2016, 4 (4), 39. https://doi.org/10.3390/dj4040039 4. Ruggiero, S. L.; Dodson, T. B.; Fantasia, J.; Goodday, R.; Aghaloo, T.; Mehrotra, B.; O'Ryan, F.; American Association of, O.; Maxillofacial, S., American Association of Oral and Maxillofacial Surgeons position paper on medication-related osteonecrosis of the jaw--2014 update. J. Oral Maxillofac. Surg. 2014, 72 (10), 1938-56. https://doi.org/10.1016/j.joms.2014.04.031 16


Page 16 of 36

Page 17 of 36 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


5. Sanchis, J. M.; Bagán, J. V.; Murillo, J.; Díaz, J. M.; Asensio, L., Risk of developing BRONJ among patients exposed to intravenous bisphosphonates following tooth extraction. Quintessence Int. 2014, 45 (9), 769-777. https://doi.org/10.3290/j.qi.a32243 6. Assael, L. A., Oral Bisphosphonates as a Cause of Bisphosphonate-Related Osteonecrosis of the Jaws: Clinical Findings, Assessment of Risks, and Preventive Strategies. J. Oral Maxillofac. Surg. 2009, 67 (5), 35-43. https://doi.org/10.1016/j.joms.2009.01.003 7. Vescovi, P.; Nammour, S., Bisphosphonate-Related Osteonecrosis of the Jaw (BRONJ) therapy. A critical review. Minerva Stomatol. 2010, 59 (4), 181-203. 8. Paulo, S.; Abrantes, A. M.; Laranjo, M.; Carvalho, L.; Serra, A.; Botelho, M. F.; Ferreira, M. M., Bisphosphonate-related osteonecrosis of the jaw: specificities. Oncol. Rev. 2014, 8 (2), 254. https://doi.org/10.4081/oncol.2014.254 9. Marx, R. E., A decade of bisphosphonate bone complications: what it has taught us about bone physiology. Int. J. Oral Maxillofac. Implants 2012, 29 (29), e247-e258. https://doi.org/10.11607/jomi.te61 10. Hansen, P. J.; Knitschke, M.; Draenert, F. G.; Irle, S.; Neff, A., Incidence of bisphosphonate-related osteonecrosis of the jaws (BRONJ) in patients taking bisphosphonates for osteoporosis treatment - a grossly underestimated risk? Clin. Oral Investig. 2013, 17 (8), 1829-1837. https://doi.org/10.1007/s00784-012-0873-3 11. Odvina, C. V.; Zerwekh, J. E.; D Sudhaker, R.; Naim, M.; Gottschalk, F. A.; Pak, C. Y. C., Severely suppressed bone turnover: a potential complication of alendronate therapy. J.clin.endocrinol.metab 2005, 90 (3), 1294-301. https://doi.org/10.1210/jc.2004-0952 12. Idris, A.; Rojas, J.; Greig, I.; Van'T-Hof, R.; Ralston, S., Aminobisphosphonates cause osteoblast apoptosis and inhibit bone nodule formation in vitro. Calcif. Tissue Int. 2008, 82 (3), 191. https://doi.org/10.1007/s00223-008-9104-y 13. Abe, K.; Yoshimura, Y.; Deyama, Y.; Kikuiri, T.; Hasegawa, T.; Tei, K.; Shinoda, H.; Suzuki, K.; Kitagawa, Y., Effects of bisphosphonates on osteoclastogenesis in RAW264.7 cells. Int. J. Mol. Med. 2012, 29 (6), 1007-1015. https://doi.org/10.3892/ijmm.2012.952 14. Ogata, K.; Katagiri, W.; Osugi, M.; Kawai, T.; Sugimura, Y.; Hibi, H.; Nakamura, S.; Ueda, M., Evaluation of the therapeutic effects of conditioned media from mesenchymal stem cells in a rat bisphosphonate-related osteonecrosis of the jaw-like model. Bone 2015, 74, 95-105. https://doi.org/10.1016/j.bone.2015.01.011 15. He, L. H.; Xiao, E.; An, J. G.; He, Y.; Chen, S.; Zhao, L.; Zhang, T.; Zhang, Y., Role of Bone Marrow Stromal Cells in Impaired Bone Repair from BRONJ Osseous Lesions. J. Dent. Res. 2017, 96 (5), 539-546. https://doi.org/10.1177/0022034517691507 16. Hughes, D. E.; Wright, K. R.; Uy, H. L.; Sasaki, A.; Yoneda, T.; Roodman, G. D.; Mundy, G. R.; Boyce, B. F., Bisphosphonates promote apoptosis in murine osteoclasts in vitro and in vivo. J. Bone Miner. Res. 2010, 10 (10), 1478-1487. https://doi.org/10.1002/jbmr.5650101008 17. Verena, S.; Fournier, P. G.; Akeila, B.; Ismahène, B. D.; Hannu, M. N. N.; Marc, C.; F Hal, E.; Vincent, C.; Philippe, C., Nitrogen-containing bisphosphonates can inhibit angiogenesis in vivo without the involvement of farnesyl pyrophosphate synthase. Bone 2011, 48 (2), 259-266. https://doi.org/10.1016/j.bone.2010.09.035 18. Black, D. M.; Delmas, P. D.; Richard, E.; Reid, I. R.; Steven, B.; Cauley, J. A.; Felicia, C.; Péter, L.; Chung, L. P.; Zulema, M., Once-yearly zoledronic acid for treatment of postmenopausal osteoporosis. N. Engl. J. Med. 2007, 356 (18), 1809-1822. 17



https://doi.org/10.1056/NEJMoa067312 19. Kostantinos, Z.; Eufimia, B.; Pavlos, Z.; Ellada, E.; Theodore, K.; Hellie, L.; George, T.; Ioannis, K.; Karamanos, N. K., The impact of zoledronic acid therapy in survival of lung cancer patients with bone metastasis. Int. J. Cancer 2010, 125 (7), 1705-1709. https://doi.org/10.1002/ijc.24470 20. Yoshimura, H.; Muneta, T.; Nimura, A.; Yokoyama, A.; Koga, H.; Sekiya, I., Comparison of rat mesenchymal stem cells derived from bone marrow, synovium, periosteum, adipose tissue, and muscle. Cell Tissue Res. 2007, 327 (3), 449-62. https://doi.org/10.1007/s00441-006-0308-z 21. Ando, Y.; Matsubara, K.; Ishikawa, J.; Fujio, M.; Shohara, R.; Hibi, H.; Ueda, M.; Yamamoto, A., Stem cell-conditioned medium accelerates distraction osteogenesis through multiple regenerative mechanisms. Bone 2014, 61 (4), 82-90. https://doi.org/10.1016/j.bone.2013.12.029 22. Hofer, H. R.; Tuan, R. S., Secreted trophic factors of mesenchymal stem cells support neurovascular and musculoskeletal therapies. Stem Cell. Res. Ther. 2016, 7 (1), 131. https://doi.org/10.1186/s13287-016-0394-0 23. Kikuiri, T.; Kim, I.; Yamaza, T.; Akiyama, K.; Zhang, Q.; Li, Y.; Chen, C.; Chen, W. J.; Wang, S.; Le, A. D., Cell-Based Immunotherapy With Mesenchymal Stem Cells Cures Bisphosphonate-Related Osteonecrosis of the Jaw–like Disease in Mice. J. Bone Miner. Res. 2010, 25 (7), 1668-1679. https://doi.org/10.1002/jbmr.37 24. Barba-Recreo, P.; Jl, D. C. P. D. V.; Georgiev-Hristov, T.; Ruiz, B.-B. E.; Abarrategi, A.; Burgueño, M.; García-Arranz, M., Adipose-derived stem cells and platelet-rich plasma for preventive treatment of bisphosphonate-related osteonecrosis of the jaw in a murine model. J. Craniomaxillofac. Surg. 2015, 43 (7), 1161-1168. https://doi.org/10.1016/j.jcms.2015.04.026 25. Ogata, K.; Katagiri, W.; Hibi, H., Secretomes from mesenchymal stem cells participate in the regulation of osteoclastogenesis in vitro. Clin. Oral Investig. 2017, 21 (6), 1979-1988. https://doi.org/10.1007/s00784-016-1986-x 26. Takashi, K.; Insoo, K.; Takyoshi, Y.; Kentaro, A.; Qunzhou, Z.; Yunsheng, L.; Chider, C.; Wanjun, C.; Songlin, W.; Le, A. D., Cell-based immunotherapy with mesenchymal stem cells cures bisphosphonate-related osteonecrosis of the jaw-like disease in mice. J. Bone Miner. Res. 2010, 25 (7), 1668-1679. https://doi.org/10.1002/jbmr.37 27. Lombard, T.; Neirinckx, V.; Rogister, B.; Gilon, Y.; Wislet, S., MedicationRelated Osteonecrosis of the Jaw: New Insights into Molecular Mechanisms and Cellular Therapeutic Approaches. Stem Cells Int. 2016, (2016-9-18) 2016, 2016 (2), 116. https://doi.org/10.1155/2016/8768162 28. Matsuura, Y.; Atsuta, I.; Ayukawa, Y.; Yamaza, T.; Kondo, R.; Takahashi, A.; Ueda, N.; Oshiro, W.; Tsukiyama, Y.; Koyano, K., Therapeutic interactions between mesenchymal stem cells for healing medication-related osteonecrosis of the jaw. Stem Cell. Res. Ther. 2016, 7 (1), 119. https://doi.org/10.1186/s13287-016-0367-3 29. Fischer, U. M.; Harting, M. T.; Jimenez, F.; Monzon-Posadas, W. O.; Xue, H.; Savitz, S. I.; Laine, G. A.; Cox, C. S., Jr., Pulmonary passage is a major obstacle for intravenous stem cell delivery: the pulmonary first-pass effect. Stem Cells Dev 2009, 18 (5), 683-92. https://doi.org/10.1089/scd.2008.0253 30. Mandel, K.; Yang, Y.; Schambach, A.; Glage, S.; Otte, A.; Hass, R., Mesenchymal stem cells (MSC) directly interact with breast cancer cells and promote tumor cell growth in vitro and in vivo. Stem Cells Dev. 2013, 22 (23), 3114-27. https://doi.org/10.1089/scd.2013.0249 31. Ide, C.; Nakai, Y.; Nakano, N.; Seo, T. B.; Yamada, Y.; Endo, K.; Noda, T.; Saito, 18


Page 18 of 36

Page 19 of 36 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


F.; Suzuki, Y.; Fukushima, M., Bone marrow stromal cell transplantation for treatment of sub-acute spinal cord injury in the rat. Brain Res. 2010, 1332 (3), 32-47. https://doi.org/10.1016/j.brainres.2010.03.043 32. Zimmermann, C. E.; Gierloff, M.; Hedderich, J.; Aã§Il, Y.; Wiltfang, J.; Terheyden, H., Survival of transplanted rat bone marrow-derived osteogenic stem cells in vivo. Tissue Eng., Part A 2011, 17 (7-8), 1147-1156. https://doi.org/10.1089/ten.TEA.2009.0577 33. Alarçin, E.; Lee, T. Y.; Karuthedom, S.; Mohammadi, M.; Brennan, M. A.; Lee, D. H.; Marrella, A.; Zhang, J.; Syla, D.; Zhang, Y. S., Injectable shear-thinning hydrogels for delivering osteogenic and angiogenic cells and growth factors. Biomater. Sci. 2018, 6 (6), 1604-1615. https://doi.org/10.1039/c8bm00293b 34. Sarker, B.; Zehnder, T.; Rath, S. N.; Horch, R. E.; Kneser, U.; Detsch, R.; Boccaccini, A. R., Oxidized Alginate-Gelatin Hydrogel: A Favorable Matrix for Growth and Osteogenic Differentiation of Adipose-Derived Stem Cells in 3D. ACS Biomater. Sci. Eng. 2017, 3 (8), 1730-1737. https://doi.org/10.1021/acsbiomaterials.7b00188 35. Seo, B. B.; Koh, J. T.; Song, S. C., Tuning physical properties and BMP-2 release rates of injectable hydrogel systems for an optimal bone regeneration effect. Biomaterials 2017, 122, 91-104. https://doi.org/10.1016/j.biomaterials.2017.01.016 36. Huanan, W.; Qin, Z.; Boerman, O. C.; Nijhuis, A. W. G.; Jansen, J. A.; Yubao, L.; Leeuwenburgh, S. C. G., Combined delivery of BMP-2 and bFGF from nanostructured colloidal gelatin gels and its effect on bone regeneration in vivo. J. Control. Release 2013, 166 (2), 172-181. https://doi.org/10.1016/j.jconrel.2012.12.015 37. Wang, C. K.; Ho, M. L.; Wang, G. J.; Chang, J. K.; Chen, C. H.; Fu, Y. C.; Fu, H. H., Controlled-release of rhBMP-2 carriers in the regeneration of osteonecrotic bone. Biomaterials 2009, 30 (25), 4178-86. https://doi.org/10.1016/j.biomaterials.2009.04.029 38. Kuroshima, S.; Sasaki, M.; Nakajima, K.; Tamaki, S.; Hayano, H.; Sawase, T., Transplantation of Non-cultured Stromal Vascular Fraction Cells of Adipose Tissue Ameliorates Osteonecrosis of the Jaw-like Lesions in Mice. J. Bone Miner. Res. 2017, 33 (1), 154-166. https://doi.org/10.1002/jbmr.3292 39. Lotfy, A.; Salama, M.; Zahran, F.; Jones, E.; Badawy, A.; Sobh, M., Characterization of mesenchymal stem cells derived from rat bone marrow and adipose tissue: a comparative study. Int J Stem Cells 2014, 7 (2), 135-142. https://doi.org/10.15283/ijsc.2014.7.2.135 40. Yuka, I.; Kentaro, Y.; Shin-Ichiro, H.; Hiroshi, M.; Masahiro, T.; Fukka, Y.; Kiyofumi, Y.; Yoshitaka, T.; Yusuke, E.; Shigeru, N., Comparison of mesenchymal stem cells from adipose tissue and bone marrow for ischemic stroke therapy. Cytotherapy 2011, 13 (6), 675-685. https://doi.org/10.3109/14653249.2010.549122 41. Lee, E. Y.; Xia, Y.; Kim, W. S.; Kim, M. H.; Kim, T. H.; Kim, K. J.; Park, B. S.; Sung, J. H., Hypoxia-enhanced wound-healing function of adipose-derived stem cells: increase in stem cell proliferation and up-regulation of VEGF and bFGF. Wound Repair Regen. 2010, 17 (4), 540-547. https://doi.org/10.1111/j.1524-475X.2009.00499.x 42. Osmałek, T.; Froelich, A.; Tasarek, S., Application of gellan gum in pharmacy and medicine. Int. J. Pharm. 2014, 466 (1-2), 328-340. https://doi.org/10.1016/j.ijpharm.2014.03.038 43. Stevens, L. R.; Gilmore, K. J.; Wallace, G. G.; In Het Panhuis, M., Tissue engineering with gellan gum. Biomater Sci 2016, 4 (9), 1276-90. https://doi.org/10.1039/c6bm00322b 44. Zia, K. M.; Tabasum, S.; Khan, M. F.; Akram, N.; Akhter, N.; Noreen, A.; Zuber, 19



M., Recent trends on Gellan Gum blends with natural and synthetic polymers: A review. Int. J. Biol. Macromol. 2017, 109, 1068-1087. https://doi.org/10.1016/j.ijbiomac.2017.11.099 45. Smith, A. M.; Shelton, R. M.; Perrie, Y.; Harris, J. J., An initial evaluation of gellan gum as a material for tissue engineering applications. J. Biomater. Appl. 2007, 22 (3), 241-54. https://doi.org/10.1177/0885328207076522 46. Sun, X. F.; Wang, H. H.; Jing, Z. X.; Mohanathas, R., Hemicellulose-based pHsensitive and biodegradable hydrogel for controlled drug delivery. Carbohydr. Polym. 2013, 92 (2), 1357-66. https://doi.org/10.1016/j.carbpol.2012.10.032 47. Yamaguchi, T., . Inhibitory activity of heat treated vegetables and indigestible polysaccharides on mutagenicity. Mutat. Res. 1992, 284 (2), 205-213. https://doi.org/10.1016/0027-5107(92)90004-L 48. Ebringerová, A.; Hromádková, Z.; Alfödi, J.; Hřı́Balová, V., The immunologically active xylan from ultrasound-treated corn cobs: extractability, structure and properties. Carbohydr. Polym. 1998, 37 (3), 231-239. https://doi.org/10.1016/S01448617(98)00065-4 49. Morris, G. A.; Hromádková, Z.; Ebringerová, A.; Malovı́Ková, A.; Alföldi, J.; Harding, S. E., Modification of pectin with UV-absorbing substitutents and its effect on the structural and hydrodynamic properties of the water-soluble derivatives. Carbohydr. Polym. 2002, 48 (4), 351-359. https://doi.org/10.1016/S01448617(01)00268-5 50. Oliveira, E. E.; Silva, A. E.; Júnior, T. N.; Gomes, M. C. S.; Aguiar, L. M.; Marcelino, H. R.; Araújo, I. B.; Bayer, M. P.; Ricardo, N. M. P. S.; Oliveira, A. G., Xylan from corn cobs, a promising polymer for drug delivery: Production and characterization. Bioresour. Technol. 2010, 101 (14), 5402-5406. https://doi.org/10.1016/j.biortech.2010.01.137 51. Silva-Correia, J.; Oliveira, J. M.; Caridade, S. G.; Oliveira, J. T.; Sousa, R. A.; Mano, J. F.; Reis, R. L., Gellan gum-based hydrogels for intervertebral disc tissueengineering applications. J. Tissue Eng. Regen. Med. 2011, 5 (6), e97-107. https://doi.org/10.1002/term.363 52. Zeng, L.; Yao, Y.; Wang, D. A.; Chen, X., Effect of microcavitary alginate hydrogel with different pore sizes on chondrocyte culture for cartilage tissue engineering. Mater. Sci. Eng. C Mater. Biol. Appl. 2014, 34, 168-75. https://doi.org/10.1016/j.msec.2013.09.003 53. Park, H. J.; Jin, Y.; Shin, J.; Yang, K.; Lee, C.; Yang, H. S.; Cho, S. W., CatecholFunctionalized Hyaluronic Acid Hydrogels Enhance Angiogenesis and Osteogenesis of Human Adipose-Derived Stem Cells in Critical Tissue Defects. Biomacromolecules 2016, 17 (6), 1939-1948. https://doi.org/10.1021/acs.biomac.5b01670 54. Kim, S. H.; Moon, H. H.; Kim, H. A.; Hwang, K. C.; Lee, M.; Choi, D., Hypoxiainducible vascular endothelial growth factor-engineered mesenchymal stem cells prevent myocardial ischemic injury. Mol. Ther. 2011, 19 (4), 741-50. https://doi.org/10.1038/mt.2010.301 55. Kaibuchi, N.; Iwata, T.; Yamato, M.; Okano, T.; Ando, T., Multipotent mesenchymal stromal cell sheet therapy for bisphosphonate-related osteonecrosis of the jaw in a rat model. Acta Biomater. 2016, 42, 400-410. https://doi.org/10.1016/j.actbio.2016.06.022 56. Birnbaum, R. S.; Bowsher, R. R.; Wiren, K. M., Changes in IGF-I and -II expression and secretion during the proliferation and differentiation of normal rat osteoblasts. J. Endocrinol. 1995, 144 (2), 251. https://doi.org/10.1677/joe.0.1440251 57. Jacobo, S. M.; Kazlauskas, A., Insulin-like growth factor 1 (IGF-1) stabilizes 20


Page 20 of 36

Page 21 of 36 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


nascent blood vessels. J. Biol. Chem. 2015, 290 (10), 6349-60. https://doi.org/10.1074/jbc.M114.634154 58. Bikle, D. D.; Wang, Y., Insulin-like Growth Factor-I and Bone. Ibms Bonekey 2011, 8 (7), 328-341. https://doi.org/10.1138/20110521 59. Bermudez, M. A.; Sendonlago, J.; Eiro, N.; Treviño, M.; Gonzalez, F.; Yebrapimentel, E.; Giraldez, M. J.; Macia, M.; Lamelas, M. L.; Saa, J., Corneal epithelial wound healing and bactericidal effect of conditioned medium from human uterine cervical stem cells. Invest. Ophthalmol. Vis. Sci. 2015, 56 (2), 983-92. https://doi.org/10.1167/iovs.14-15859 60. Korsak, J.; Rzeszotarska, A.; Marczyński, W.; Jabłońska, I.; Białecki, J.; Walczak, P., Concentration of platelet derived-growth factors in concentrates used to regenerate injured bone tissue. Ortop Traumatol Rehabil 2013, 15 (15), 379-388. https://doi.org/10.5604/15093492.1084239 61. Wu, Q.; Zhou, X.; Huang, D.; Ji, Y.; Kang, F., IL-6 Enhances Osteocyte-Mediated Osteoclastogenesis by Promoting JAK2 and RANKL Activity In Vitro. Cell. Physiol. Biochem. 2017, 41 (4), 1360. https://doi.org/10.1159/000465455 62. Naito, H.; Yoshimura, M.; Mizuno, T.; Takasawa, S.; Tojo, T.; Taniguchi, S., The advantages of three-dimensional culture in a collagen hydrogel for stem cell differentiation. J. Biomed. Mater. Res., Part A 2013, 101 (10), 2838-2845. https://doi.org/10.1002/jbm.a.34578 63. Zhao, X.; Liu, S.; Yildirimer, L.; Zhao, H.; Ding, R.; Wang, H.; Cui, W.; Weitz, D., Microfluidics‐Assisted Osteogenesis: Injectable Stem Cell‐Laden Photocrosslinkable Microspheres Fabricated Using Microfluidics for Rapid Generation of Osteogenic Tissue Constructs. Adv. Funct. Mater. 2016, 26 (17), 2976-2976. https://doi.org/10.1002/adfm.201670110 64. Lopez-Cebral, R.; Civantos, A.; Ramos, V.; Seijo, B.; Lopez-Lacomba, J. L.; SanzCasado, J. V.; Sanchez, A., Gellan gum based physical hydrogels incorporating highly valuable endogen molecules and associating BMP-2 as bone formation platforms. Carbohydr. Polym. 2017, 167, 345-355. https://doi.org/10.1016/j.carbpol.2017.03.049 65. Lysdahl, H.; Baatrup, A.; Foldager, C. B.; Bunger, C., Preconditioning Human Mesenchymal Stem Cells with a Low Concentration of BMP2 Stimulates Proliferation and Osteogenic Differentiation In Vitro. BioRes. Open Access 2014, 3 (6), 278-85. https://doi.org/10.1089/biores.2014.0044 66. Kempen, D. H.; Lu, L.; Hefferan, T. E.; Creemers, L. B.; Maran, A.; Classic, K. L.; Dhert, W. J.; Yaszemski, M. J., Retention of in vitro and in vivo BMP-2 bioactivities in sustained delivery vehicles for bone tissue engineering. Biomaterials 2008, 29 (22), 3245-52. https://doi.org/10.1016/j.biomaterials.2008.04.031 67. Usui, M.; Xing, L.; Drissi, H.; Zuscik, M.; O'Keefe, R.; Chen, D.; Boyce, B. F., Murine and chicken chondrocytes regulate osteoclastogenesis by producing RANKL in response to BMP2. J. Bone Miner. Res. 2008, 23 (3), 314-25. https://doi.org/10.1359/jbmr.071025 68. Tsubaki, M.; Satou, T.; Itoh, T.; Imano, M.; Yanae, M.; Kato, C.; Takagoshi, R.; Komai, M.; Nishida, S., Bisphosphonate- and statin-induced enhancement of OPG expression and inhibition of CD9, M-CSF, and RANKL expressions via inhibition of the Ras/MEK/ERK pathway and activation of p38MAPK in mouse bone marrow stromal cell line ST2. Mol. Cell. Endocrinol. 2012, 361 (1-2), 219-231. https://doi.org/10.1016/j.mce.2012.05.002 69. Cerqueira, M. T.; da Silva, L. P.; Santos, T. C.; Pirraco, R. P.; Correlo, V. M.; Reis, R. L.; Marques, A. P., Gellan gum-hyaluronic acid spongy-like hydrogels and cells from adipose tissue synergize promoting neoskin vascularization. ACS Appl. Mater. 21



Interfaces 2014, 6 (22), 19668-79. https://doi.org/10.1021/am504520j 70. Daly, A. C.; Pitacco, P.; Nulty, J.; Cunniffe, G. M.; Kelly, D. J., 3D printed microchannel networks to direct vascularisation during endochondral bone repair. Biomaterials 2018, 162, 34-46. https://doi.org/10.1016/j.biomaterials.2018.01.057 71. Li, Y.; Xu, J.; Mao, L.; Liu, Y.; Gao, R.; Zheng, Z.; Chen, W.; Le, A.; Shi, S.; Wang, S., Allogeneic Mesenchymal Stem Cell Therapy for Bisphosphonate-Related Jaw Osteonecrosis in Swine. Stem Cells Dev. 2013, 22 (14), 2047. https://doi.org/10.1089/scd.2012.0615

22


Page 22 of 36

Page 23 of 36 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


Table 1. Primer pairs used in q-PCR analysis Gene Forward primer COL Ia1 5’GCCTCCCA GAACATCAC CTA3’ ALP 5’AACGTGGC CAAGAACAT CATCA 3’ VEGF 5’GTACCTCC ACCATGCCA AGT 3’ OCN 5’GACTCTGA GTCTGACAA A 3’ Runx2 5’CCATAACG GTCTTCACA AATCCT3’ RANKL 5’GTCGTTAA AACCAGCAT C3’ OPG 5’CGAAGAGG CATTCTTCA G3’ GAPDH GGCACAGTC AAGGCTGAG AATG Figure 1

Reverse primer 5’GCAGGGACT TCTTGAGGTTG 3’ 5’TGTCCATCTC CAGCCGTGTC 3’ 5’AGGCAGCAA GAGAGATTGG TCACT 3’ 5’AGTCCATTGT TGAGGTAG 3’ 5’TCTGTCTGTG CCTTCTTGGTT C 3’ 5’CCTGACCAG TTCTTAGTG3’ 5’TCTGCATTCA CTTTGGTC3’ ATGGTGGTGA AGACGCCAGT A

Physical and

mechanical characterization of LAGG and LAGG-PM hydrogels. (A) Gel state of LAGG and LAGG-PM before and after gelation. (B) Gelation times required for formation of LAGG and LAGG-PM hydrogels (n = 3, **p < 0.01). (C) FTIR spectra of LAGG and LAGG–PM powders. (D) SEM images showing interconnected pores of LAGG and LAGG-PM hydrogels (scale bar = 10 μm). (E) Internal pore size (n = 8). (F) Swelling properties were measured after incubation at 37° (n = 3). (G) Weight loss of LAGG and LAGG-PM hydrogels soaked in PBS at 37° (n=3). (H) The storage modulus (G′) and loss modulus (G″) of LAGG and LAGG-PM hydrogels were measured by rheometric. (I) The average elastic moduli of LAGG and LAGG-PM hydrogels at 1 Hz (n = 3, **p < 0.01).

23



Figure 2 Cell-hydrogel 3D coculture (A)Flow cytometry analysis of extracted rADSCs. (B) Microscope image showing spreading and morphology of rADSCs in 3D culture after 14 days (scale bar=250 µm). (C) Live (green) and dead (red) fluorescence microscopy images of rADSCs inside 3D LAGG and LAGG-PM hydrogels during the 3D culture period (scale bar=250 µm). Live (green) cells were labelled with calcein AM and dead (red) cells were labelled with ethidium homodimer-1. (D) Cell viability detected by CCK8 assay (n = 4, *p < 0.05, **p < 0.01). (E) Quantification for percentage of live cell. (F-H) The level of VEGF, IGF-1 and RANKL secreted from rADSCs were determined by ELISA quantification (n = 4, *p < 0.05, **p < 0.01). (I) Osteoclast differentiation of RAW264.7 cells induced by conditioned medium (scale bar=100 µm). (J) Quantification for number of osteoclasts per high power field (X200) (n = 4, **p < 0.01).

24


Page 24 of 36

Page 25 of 36 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 3 Differentiation of rADSCs within 3D LAGG-PM hydrogels and BMP-2 release in vitro. (A, B) mRNA expressions of COL Ia1 and ALP with BMP-2 of different concentration (n = 3, ◆significant differences between 0 and 25 ~ 200 ng/ml; *significant differences between 25 ~ 200 ng/ml, *p < 0.05, **p < 0.01). (C) BMP-2 of 50 ng/ml cumulatively released from LAGG and LAGG-PM hydrogels over time. (D-J) mRNA expressions of COL Ia1(D), ALP(E), RUNX-2(F), OCN(G), VEGF(H), RANKL(I), OPG(J) and RANKL/OPG ratio(n = 3, *p < 0.05, **p < 0.01).

25



Figure 4 Establishment of a MRONJ-like model in SD rats. (A) Timeline design of experimental procedures in this study. (B) The incidence of MRONJ-like disease was shown as the percentage of exposed bones in BP-treated rats, whereas rats with untreated as controls. (C) Clinical appearance of necrotic bone and mucosal healing. The red circles represented images of the mucosa at the extraction site. (D) Micro-CT analysis showed reduced bone formation and complete healing of bone tissue from various sections (scale bar=1 mm). (E) Circle a cylinder through the white circle along the alveolar socket as Volume of Interest (VOI) (scale bar=1 mm). (F, G) Bone volume fraction(BV/TV) and bone mineral density(BMD) in VOI (n = 4, **p < 0.01). (H, I) Histological images revealed the absence of an epithelial lining in the extraction socket (black dot line), and healed mucosa with complete epithelial coverage (black arrow) (scale bar=100 µm). NB = necrotic bone, B = newly formed bone, IF=inflammatory infiltration, E=epithelium, CT=connective tissues. (J) Masson’s trichrome staining showed necrotic bone (NB) and newly formed bone (B) at the extraction site (scale bar=100 µm). (L) The percentage of empty osteocyte lacunae counted in the BPtreatment group was significantly increased (n = 4, **p < 0.01). (K, M) TRAP staining showed significant reduction in the average number of osteoclasts (black arrow) per high power field (X200) in the BP treated group (scale bar=100 µm, n = 4, **p < 0.01).

25


Page 26 of 36

Page 27 of 36 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. The therapeutic effects of the LAGG-PM hydrogel loaded with rADSCs in a rat model of MRONJ after 4 weeks. (A) The clinical appearance after 4 weeks of treatment. The red circles indicate images of the mucosal healing at the extraction site. (B) The percentage of exposed bone and mucosal healing after 4 weeks of treatment is shown. (C) Micro-CT analysis showed the healing of the bone tissue at the extraction site after treatment from various sections (scale bar=1 mm). (D, E) Bone volume fraction(BV/TV) and bone mineral density(BMD) in VOI (n = 4, *p < 0.05, **p < 0.01).(F-I) HE staining and Masson’s trichrome staining showed mucosal healing (black arrow) and bone regeneration (scale bar=100 µm). NB = necrotic bone, B = newly formed bone, IF=inflammatory infiltration, E=epithelium, CT=connective tissues, Green arrow represented LAGG-PM hydrogel. (K) The percentage of empty osteocyte lacunae that were counted (n = 4, ◆significant differences between Control and treatment groups; *significant differences between treatment groups; *p < 0.05, **p < 0.01). (J, L) TRAP staining showed the average number of osteoclasts (black arrow) per high-power field (X200, scale bar=100 µm, n = 4, *p < 0.05, **p < 0.01).

27



Figure 6 Sequential fluorescence image analysis of LAGG-PM hydrogel loaded with rADSCs in a rat model of MRONJ after 8 weeks. (A)The sequential fluorescent labelling analysis of newly formed bone at the extraction site, labelled by tetracycline hydrochloride (yellow), calcein (green), and alizarin red (red) (scale bar=1000 μm, scale bar=200 μm). (B, C, D) The newly formed bone was quantified by calculating the percentage of area of fluorochrome stained bone after 2, 4, 7 weeks (n = 4, ◆significant differences between Control and treatment groups, *significant differences between treatment groups, *p < 0.05, **p < 0.01).

27


Page 28 of 36

Page 29 of 36 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

Microfiber-reinforced composite hydrogels loaded with rat adiposederived stem cells and BMP-2 for the treatment of medication-related osteonecrosis of the jaw in a rat model Haoran Ning†, Xiaowei Wu†,‡, Qing Wu†, Wanlu Yu†, Huaiji Wang†, Shang Zheng†, Yunong Chen†, Yongyong Li*,§, Jiansheng Su*,†

29



Table of Contents Graphic 307x170mm (150 x 150 DPI)


Page 30 of 36

Page 31 of 36 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 Physical and mechanical characterization of LAGG and LAGG-PM hydrogels. (A) Gel state of LAGG and LAGG-PM before and after gelation. (B) Gelation times required for formation of LAGG and LAGG-PM hydrogels (n = 3, **p < 0.01). (C) FTIR spectra of LAGG and LAGG–PM powders. (D) SEM images showing interconnected pores of LAGG and LAGG-PM hydrogels (scale bar = 10 μm). (E) Internal pore size (n = 8). (F) Swelling properties were measured after incubation at 37° (n = 3). (G) Weight loss of LAGG and LAGGPM hydrogels soaked in PBS at 37° (n=3). (H) The storage modulus (G′) and loss modulus (G″) of LAGG and LAGG-PM hydrogels were measured by rheometric. (I) The average elastic moduli of LAGG and LAGGPM hydrogels at 1 Hz (n = 3, **p < 0.01). 177x80mm (300 x 300 DPI)



Figure 2 Cell-hydrogel 3D coculture (A)Flow cytometry analysis of extracted rADSCs. (B) Microscope image showing spreading and morphology of rADSCs in 3D culture after 14 days (scale bar=250 µm). (C) Live (green) and dead (red) fluorescence microscopy images of rADSCs inside 3D LAGG and LAGG-PM hydrogels during the 3D culture period (scale bar=250 µm). Live (green) cells were labelled with calcein AM and dead (red) cells were labelled with ethidium homodimer-1. (D) Cell viability detected by CCK8 assay (n = 4, *p < 0.05, **p < 0.01). (E) Quantification for percentage of live cell. (F-H) The level of VEGF, IGF-1 and RANKL secreted from rADSCs were determined by ELISA quantification (n = 4, *p < 0.05, **p < 0.01). (I) Osteoclast differentiation of RAW264.7 cells induced by conditioned medium (scale bar=100 µm). (J) Quantification for number of osteoclasts per high power field (X200) (n = 4, **p < 0.01). 339x218mm (150 x 150 DPI)


Page 32 of 36

Page 33 of 36 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 3 Differentiation of rADSCs within 3D LAGG-PM hydrogels and BMP-2 release in vitro. (A, B) mRNA expressions of COL Ia1 and ALP with BMP-2 of different concentration (n = 3, ◆significant differences between 0 and 25 ~ 200 ng/ml; *significant differences between 25 ~ 200 ng/ml, *p < 0.05, **p < 0.01). (C) BMP-2 of 50 ng/ml cumulatively released from LAGG and LAGG-PM hydrogels over time. (D-J) mRNA expressions of COL Ia1(D), ALP(E), RUNX-2(F), OCN(G), VEGF(H), RANKL(I), OPG(J) and RANKL/OPG ratio(n = 3, *p < 0.05, **p < 0.01) 329x187mm (150 x 150 DPI)



Figure 4 Establishment of a MRONJ-like model in SD rats. (A) Timeline design of experimental procedures in this study. (B) The incidence of MRONJ-like disease was shown as the percentage of exposed bones in BPtreated rats, whereas rats with untreated as controls. (C) Clinical appearance of necrotic bone and mucosal healing. The red circles represented images of the mucosa at the extraction site. (D) Micro-CT analysis showed reduced bone formation and complete healing of bone tissue from various sections (scale bar=1 mm). (E) Circle a cylinder through the white circle along the alveolar socket as Volume of Interest (VOI) (scale bar=1 mm). (F, G) Bone volume fraction(BV/TV) and bone mineral density(BMD) in VOI (n = 4, **p < 0.01). (H, I) Histological images revealed the absence of an epithelial lining in the extraction socket (black dot line), and healed mucosa with complete epithelial coverage (black arrow) (scale bar=100 µm). NB = necrotic bone, B = newly formed bone, IF=inflammatory infiltration, E=epithelium, CT=connective tissues. (J) Masson’s trichrome staining showed necrotic bone (NB) and newly formed bone (B) at the extraction site (scale bar=100 µm). (L) The percentage of empty osteocyte lacunae counted in the BP-treatment group was significantly increased (n = 4, **p < 0.01). (K, M) TRAP staining showed significant reduction in the average number of osteoclasts (black arrow) per high power field (X200) in the BP treated group (scale bar=100 µm, n = 4, **p < 0.01) 326x325mm (150 x 150 DPI)


Page 34 of 36

Page 35 of 36 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. The therapeutic effects of the LAGG-PM hydrogel loaded with rADSCs in a rat model of MRONJ after 4 weeks. (A) The clinical appearance after 4 weeks of treatment. The red circles indicate images of the mucosal healing at the extraction site. (B) The percentage of exposed bone and mucosal healing after 4 weeks of treatment is shown. (C) Micro-CT analysis showed the healing of the bone tissue at the extraction site after treatment from various sections (scale bar=1 mm). (D, E) Bone volume fraction(BV/TV) and bone mineral density(BMD) in VOI (n = 4, *p < 0.05, **p < 0.01).(F-I) HE staining and Masson’s trichrome staining showed mucosal healing (black arrow) and bone regeneration (scale bar=100 µm). NB = necrotic bone, B = newly formed bone, IF=inflammatory infiltration, E=epithelium, CT=connective tissues, Green arrow represented LAGG-PM hydrogel. (K) The percentage of empty osteocyte lacunae that were counted (n = 4, ◆significant differences between Control and treatment groups; *significant differences between treatment groups; *p < 0.05, **p < 0.01). (J, L) TRAP staining showed the average number of osteoclasts (black arrow) per high-power field (X200, scale bar=100 µm, n = 4, *p < 0.05, **p < 0.01). 410x330mm (150 x 150 DPI)



Figure 6 Sequential fluorescence image analysis of LAGG-PM hydrogel loaded with rADSCs in a rat model of MRONJ after 8 weeks. (A)The sequential fluorescent labelling analysis of newly formed bone at the extraction site, labelled by tetracycline hydrochloride (yellow), calcein (green), and alizarin red (red) (scale bar=1000 μm, scale bar=200 μm). (B, C, D) The newly formed bone was quantified by calculating the percentage of area of fluorochrome stained bone after 2, 4, 7 weeks (n = 4, ◆significant differences between Control and treatment groups, *significant differences between treatment groups, *p < 0.05, **p < 0.01).


Page 36 of 36

Microfiber-reinforced composite hydrogels loaded with rat adipose

Recommend Documents