Biomimetic Gelatin Methacrylamide Hydrogel Scaffolds For Bone Tissue Engineering

Background:Bone injures and defects are serious health problems, especially those caused by complex breaks and pathological fractures arising from malformation, osteoporosis, and tumors have resulted in hundreds of millions of surgical procedures each year around the word. Autografts, allografts, and various kinds of artificial materials such as polymers, inorganic nonmetallic materials, metallic materials and their composites have been used for bone repairing or replacing. However, none of them can considered to be more economical and effective to help in treating the patients.Bone tissue engineering has emerged as an interdisciplinary field with considerable potential of development. Using new knowledge-based and cell-friendly materials that are capable of mimicking the structural, mechanical, and biological properties of natural bone holds promise in providing improved clinical therapy. Scaffold, cells, and growth factors are three important "elements of bone tissue engineering". As a hybrid constructs, scaffold with special spatially pattern plays a critical role in bone tissue engineering. Mimicking the structure and composition of bone tissues, also called the biomimetic materials, has been studied extensively over the past several decades and has attracted significant attention so far. Biomimetic materials focus on providing a 3D environment for cells proliferation and differentiation.3D environment can improve cell-cell contact, cell-matrix interactions, and increase perfusion of soluble signaling molecules, nutrients, and metabolic waste removal.Bone tissue is composed of bone extracellular matrix (ECM) and cells. Bone extracellular matrix which is primarily depended on type Ⅰ collagen is fundamental in mineral phase deposition, osteoconductive, and osteoinductive. Gelatin is a proteinaceous material obtained of collagen by hydrolysis and has almost identical fundamental molecular unit of collagen. Over the years much work has been in studying gelatin-based hydrogels that mimic biological extracellular matrix (ECM). Gelatin-based hydrogel is very attractive since it is affordability, biodegradability, good biocompatibility and easy to modify. Gelatin methacrylamide (GelMA) is an ideal physicochemical mimetic of natural extracellular matrix (ECM), making it a suitable candidate. By adding methacrylate groups to the amine-containing side-groups of gelatin and then enabling chemical cross-linking, GelMA can be stabilized at body temperature (37℃), enhance cell viability, and limit encapsulated cell elongation. Cartilage regeneration, functional vascular networks generated have been studied in previous researches. However, no one has looked bone regeneration by GelMA so far. Traditional GelMA scaffolds maybe is limited by its smooth pore wall morphology which against the natural bone morphology.Adipose-derived stem cells (ADSC) have generated considerable interest as candidates for bone tissue engineering. Studies have shown that many functions of ADSCs are similar to bone mesenchymal stem cells (BMSCs). While ADSCs have several advantages over BMSCs, including (ⅰ) abundance, (ⅱ) higher accessibility, (ⅲ) lower donor-site morbidity and (ⅳ) better angiogensis/osteogenic properties. Furthermore, it is showed that decellularized bone allografts could stimulate osteogenic differentiation of ADSCs in vitro. Recently, it is reported that osteogenic differentiated ADSCs when seeded on nano-structured bioceramic surface could promote osteogenesis.The objective of this study was to manufacture and characterize a biomimetic GelMA (Bio-GelMA) scaffold which is able to facilitate the proliferation and differentiation of ADSCs as a potential robust substitute for bone defect. The Bio-GelMA scaffolds with well-defined rough surface and porous-wall morphology were prepared by using a thermally induced phase separation (TIPS). Then we examined in vitro bone matrix formation by ADSCs cultured in biomimetic GelMA scaffolds and subsequently in vivo bone formation in an ectopic nude mouse model and a Critical-sized calvarial bone defects rat model.Our main contents include:Three parts of our research.Part one:Biomimetic gelatin methacrylamide hydrogel scaffolds preparation and characterizationObjective:Three-dimensional Bio-GelMA hydrogel scaffolds with well-defined rough surface and porous-wall morphology were prepared by using a thermally induced phase separation (TIPS).Methods:The synthesize method of GelMA:gelatin was mixed at 10%(w/v) into double distilled water at 50℃ and stirred to fully dissolved. Methacrylic anhydride (MA) was added to the gelatin solution and then allowed to react for 3h at 50℃. The mixture solution was dialyzed and filtered through neutral filter paper. Finally, the mixture was frozen at -80℃, lyophilized, and stored at room temperature. The photo-cross-linking reaction was monitored by 1H NMR.Three-dimensional GelMA scaffolds were fabricated by using a TIPS technique: To begin with, Freeze dried GelMA macromer (1.25g) was dissolved into 25 ml of double distilled water at 40℃ containing 1%(w/v) ammonium persulfate (APS) and 0.2%(v/v) N,N,N’,N-tetramethylethylenediamine (TEMED) as a redox initiator system. The mixture was incubated at -20℃ for 12h and at -80℃ for 48h. After gelation, the swollen hydrogel was cut with a self-made punch to obtain samples of equal size (length,10mm; width,8mm; and thickness about 1.5mm). And then peeled off the surface skin of the materials by freezing microtome. Finally, the hydrogel sheet was lyophilized. The structure of the freeze-dried hydrogel scaffolds were imaged using a scanning electron microscope.Results:The spectrum of gelatin methacrylamide confirms incorporation of the acrylamide double bonds at 5.3 and 5.6 ppm. The degree of the methacrylamide was about 53%.The structures of scaffolds were evaluated by SEM. The biomimetic GelMA scaffolds had low densities, high porosities, and interconnected macropores. The porosity of biomimetic GelMA scaffolds was in the order of about 85%, calculated by Image J software. And biomimetic GelMA scaffolds revealed pores approximately 100-500 nm. More importantly, the walls of scaffolds were high porous, interconnected macropores, and rough surface, compared to traditional GelMA scaffolds had a smooth surface wall.Conclusion:We have fabricated the scaffolds to mimic both physical architecture and chemical composition of natural bone ECM by thermally induced phase separation techniques.Part two:In vitro osteogenic differentiation of ADSCs in Biomimetic gelatin methacrylamide hydrogel scaffolds Objective:To study osteogenic differentiation of ADSCs in biomimetic gelatin methacrylamide hydrogel scaffolds in vitro. Biocompatibility, alkaline phosphatase (ALP) activity, mineralization at different time points and the level of genes expression were observed after ADSCs seeded in materials with osteogenic medium.Methods:Cells cultured in hydrogels with osteogenic medium were examined on days 7, 14, and 21. The excitation/emission filters were set at 488/530 nm to observe living (green) cells and at 530/580 nm to detect dead (red) cells. The number of live and dead cells from 6 samples per time point was measured by Image J software.At 7,14, and 21 days after seeding with or without scaffolds, the samples were washed for three times with PBS. ADSCs culture in 60mm cell culture dish lysed with Lysis Buffer on ice and seed in hydrogels were ground using a Potter homogeniser after the incubation with Lysis Buffer. Followed the protocol (ALP activity assay Kit), the resulting absorbance at 405 nm using an microplate reader (ThermoFisher, American).Mineralization in osteoblast cultures can be analyzed both qualitatively and quantitatively by using Alizarin red staining (ARS). On days 7,14, and 21, the samples, ADSCs seeded with or without scaffolds, was washed thrice with PBS and fixed in 4% paraformaldehyde for 1 h. The samples then washed twice with D-PBS and stained with 0.1% ARS for 15min at room temperature. These structures were then rinsed with D-PBS and observed under inverted optical microscope and images were taken using image software. To quantify matrix mineralization, the samples were incubated in 100 mM cetylpyridinium chloride for 30min to solubilize. The absorbance of the collected dye was then measured at 562 nm in Multiscan Go microplate reader.The levels of important bone-related genes were determined by RT-PCR. Total RNA was isolated using RNA extraction kit and purified according to manufactures instruction. First strand cDNAs were generated by a reverse transcriptase of 1 mg total RNAs with RevertAid First Strand cDNA Synthesis Kit. Real-time PCR was performed in triplicate with an ABI StepOne Plus system.Results:We observed that a lot of ADSCs grow in the scaffolds.As time goes on, the ADSCs and scaffolds were more and more close, in which there was apparent alignment along the scaffolds. Moreover, the ADSCs adhered the skeleton of scaffolds more tightly over time and grown along the wall of scaffolds.ALP activity quantitative analysis showed that ALP activity for the cells cultured on scaffolds was significantly higher than that on scaffolds-alone (control) at days 14 and 21 (p<0.01), except for these at day 7 (p>0.05). Besides, approximately 1 fold increase was observed at day 14 and 21. Moreover, the ALP activity of the cells cultured on scaffolds was increased over time, while the control group has the contrary results.The amount of minerals deposited significantly increased over time on ADSCs cultured on scaffolds as compared with ADSCs alone. Moreover, the mineral deposition apparently grown along the walls of scaffolds over time. Importantly, the mineral deposition quantitative analysis also showed the same results.ALP, BSP, OCN, Col I, and OPN gene expression was decreased in the ADSCs cultured on scaffolds compared with control group at day 7, while enhanced expression of OSX and RUX2 were observed. Moreover, as for ALP, OSX, OCN, and RUX2, there were no significantly different at days 14 and 21. The expression of BSP was down-regulated for ADSCs cultured on scaffolds as compared control group with day 21, except day 14. The scaffolds could also decreased the expression of Col I and OPN as compared control group with day 14, but reversal at day 21.Conclusions:The biomimetic gelatin methacrylamide hydrogel scaffolds improved promoted cell attachment, spreading, proliferation and osteogenic differentiation of rat ADSCs in vitro.Part three:In vivo osteogenic differentiation of ADSCs in biomimetic gelatin methacrylamide hydrogel scaffoldsObjective:To study osteogenic differentiation of ADSCs in biomimetic gelatin methacrylamide hydrogel scaffolds in vivo. The effect of ectopic bone formation in the subcutaneous of nude mice and repairmen of critical-size rat calvarial bone defect were observed after ADSCs seeded in materials with osteogenic medium.Methods:Each nine nude mice with 2 pockets was implanted 2 implants:Hydrogel uncoated with cells incubation with osteogenic medium for 21 days (control Group); Hydrogel coated with cells grown in osteogenic medium for 21 days (experiment Group). Four weeks after the implantation, the mice were euthanized, and then the implants were retrieved. Some implants were fixed, decalcified, dehydrated through a series of graded ethanol, embedded in paraffin, cut and stained with hematoxylin, eosin (H&E). The others were examined for H&E and immunofluorescence targeting RUX2, collagen 1, osteocalcin, osteopontin after frozen section.Adult male Sprague-Dawley rats were used in this study. The surgical site hair was cut and sterilized by iodophor. The dorsal bone on both sides of the midsagittal suture were drilled carefully by trephine, and then two Critical-sized calvarial bone defects (diameter,5.0mm) were created. All scaffolds containing cells or not were cultured in osteogenic medium for 21 days prior to implantation as per the conditions described previously. The cylindric samples were then planted and the incisions were closed with surgical sutures.At 12 weeks post-implantation, the rats were euthanized. The calvarial bones were resected, trimmed, and valuated using a micro-computed tomography (Micro-CT) scanner, three dimensional (3D) images of implants were reconstructed from the scans by mimics software. After the 3D visualizing process, bone volumes were measured in the region of interest from seven specimens in each group.After examining with Micro-CT, the implants were decalcified with Perenyi’s solution, dehydrated through a series of graded ethanol, embedded in paraffin, and cut. The sections were treated with hematoxylin/eosin (H&E) and Masson’s trichrome staining and evaluated with fluorescence microscope.Results:As shown in H&E-stained histological sections of implants -at 4 weeks after injection. In the scaffolds cultured with ADSCs groups, the scaffolds were filled with a few islands of osteoblasts, dispersed among a mass of fibrous connective tissue. Importantly, the osteoblasts, even osteoclasts, were always stuck in scaffolds. In contrast, in the scaffolds alone groups, fibrous connective tissue was also filling in the scaffolds, but the quantities were significantly decreased, and bone-liked cells were hardly seen. As shown in immunofluorescence staining images, there were significantly more CO1 I, OCN, OPN and RUX2 -positive cells were present in experiment group than in control group.The Micro-CT analysis shown that there were significantly more new bone formation was observed in experiment group as compared with control group, obtained by subtracting the bone volume of negative controls from that of the experimental constructure. H&E and MT staining also revealed increased regeneration of defective bone in the experiment group as compared with control group. Additionally, the new bone tissues were rich in osteocytes along with the scaffolds.Conclusions:In an ectopic model, the scaffolds displayed a consistently higher level of organisation with osteoblasts and highly osteogenesis-related protein expression, while we did not find any ectopic bone formation. Moreover, we don’t know the origin of the osteoblasts, whether it comes from ADSCs or nude mouse itself? But the studies also suggested that the role of the scaffolds were important to enhance the extent of cell differentiation. In a critical-size calvarial bone defect model, we found that the scaffolds with ADSCs could be observed newly bone formation, which is an amazing result and may show immense potential in designing scaffolds for bone tissue engineering.