| Background and Objectives It is the ultimate goal of periodontal treatment to achieve periodontal tissue regeneration with proper structure and function with the aid of tissue regeneration techniques. The inclusion of biomaterials in the regenerative medicine is commonly believed to maximize the beneficial effects of cellular therapies while minimizing the poor engraftment, improving persistence and controlling cell/growth factor delivery for periodontal therapies and regeneration. Hydrogels are crosslinked network of hydrophilic polymers that can swell in water to capture many times their original mass, providing a versatile platform to include desired combinations of properties for designed applications. The recently developed gelatin methacrylate(Gel MA) hydrogels have been widely used for various biomedical applications due to their suitable biological properties and tunable physical characteristics. Gel MA hydrogels closely resemble some essential properties of native extracellular matrix(ECM) due to the presence of cell attaching and matrix metalloproteinase responsive peptide motifs, which allow cells to proliferate and spread in Gel MA-based scaffolds. It can rapidly crosslink when exposed to light irradiation to form hydrogels with tunable mechanical properties. It can also be microfabricated using different methodologies including micromolding, photomasking, bioprinting, selfassembly, and microfluidic techniques to generate constructs with controlled architectures. Recent research has demonstrated the proficiency of Gel MA-based hydrogels in a wide range of tissue engineering applications including engineering of bone, cartilage, cardiac, and vascular tissues, among others. Other applications of Gel MA hydrogels, besides tissue engineering, include fundamental cell research, cell signaling, drug and gene delivery, and biosensing. Moreover, the periodontal tissue, as the tooth supporting apparatus, is located in a mechanic-active microenvironment throughout the whole life. Various studies have reported that mechanical stress has significant impacts on the physiological and pathological process of periodontium. And proper mechanical stress can promote cell viability and proliferation, while controlling the differentiation by adjusting the properties of the mechanical stimulus. Thus, a better understanding of the interaction between PDLCs and their mechanical microenvironment will facilitate treatment of periodontal diseases and promote periodontal tissue regeneration. So, this study will mainly focus on:(1) fabrication and characterization of Gel MA hydrogel, and the optimization of Gel MA physical and chemical parameters, in order to facilitate the proliferation of h PDLSCs;(2) development of customized 3D mechanical loading bio-reactor, together with the investigation of h PDLSCs viabilities and proliferations under 3D mechanical loading by this apparatus;(3) fabrication of composite hydrogel scaffold by mixing and freeze-drying of Gel MA and nano-hydroxyapatite, followed by the evaluation of their performances as regards to periondontal regeneration both in vitro and in vivo.Methods 1. Gel MA precursors were fabricated according to previous reports; surface topography was assessed by SEM, and pore size and porosity were evaluated; Young’s modulus was assessed by Instron, and swelling ratio was calculated; Gel MA hydrogel micro-array was fabricated by micro-lithgraphy. h PDLSCs was isolated and purified as previous reports, and then encapsulated in Gel MA micro-gels; the viabilities and proliferations were evaluated under different Gel MA concentrations and UV times. 2. The customized 3D mechanical loading bio-reactor was designed and fabricated by assembling a micro-motor, a linear guideway, an eccentric gear, and other parts of the apparatus. The viabilities and proliferations of h PDLSCs under 3D mechanical stimulus of different strains and frequencies applied by this apparatus were then evaluated. 3. A composite hydrogel scaffold of different concentrations of Gel MA/n HA was fabricated by freeze-drying. Their Young’s modulus was tested by Instron and pore structures were evaluated by SEM. Then, h PDLSCs were loaded onto the scaffolds, their viabilities were assessed by MTT, proliferations were tested by Brd U incorporation, and osteo-differentiations were analyzed by Alizarin Red staining and RT-PCR. After 5 w of the transplantation of h PDLSCs-laden scaffolds into nude mice, HE staining, Masson’s trichrome staining, and SEM were adopted to evaluate their periodontal regenerative capacities.Results 1. White spumescence Gel MA precursors were successfully fabricated, and can rapidly gel under photo-initiator and UV exposure to form micro-arrays. FE-SEM showed that porous inter-connected 3D network structures with smooth and uniform pores. The increasement of Gel MA concentration resulted in raised Young’s modulus increased and decreased pore size, porosity, and swelling ratio. h PDLSCs were successfully isolated from primary culture by limited diluting method, and then be encapsulated by Gel MA micro-gels. After culturing for a few days, we found that cell viability was reversely impacted as the UV times increased; however, raising Gel MA concentration seemed not relevant to cell viability. So, we chose 10% and 30 s of UV exposure to be the optimized parameters. 2. The customized 3D mechanical loading bio-reactor was successfully developed and proved to be capable of implementing 3D tensile loading on encapsulated cells with different strains and frequencies. Following experiment used h PDLSCs as the targeted cell, and results showed that 5% strain was the most appropriate strain for h PDLSCs to grow in, while frequencies seemed not a significant factor on cell viability or proliferation. 3. Gel MA/n HA composite hydrogel scaffolds with series concentrations of 5% and 10% Gel MA and 0%, 1%, 5%, and 10% n HA were successfully fabricated. In vitro experiments including SEM, MTT, Brd U, Alizarin Red staining and RT-PCR, together with in vivo experiments all agreed on that 10% Gel MA/5% n HA was the most appropriate concentration to support the viability, proliferation, and osteo-differentiation of h PDLSCs incorporated into the scaffold, and vascularization of the regenerated tissues.Conclusions The photo-sensitive Gel MA hydrogel is an ideal “soft matter”, and owns proper bio-safety, bio-compatibility, and bio-degradability, with tunable physical and chemical properties, which is capable of supporting the 3D growing of h PDLSCs. The customized 3D mechanical loading bio-reactor demonstrated accurate and easy control of loading route, disaplacement, and loading cycle, and can be properly applied in 3D cell mechanics studies, and worth further development. The Gel MA/n HA proved to be a proper composite hydrogel scaffold which is capable of supporting the osteo-differentiation of h PDLSCs, and can be considered as an alternative scaffold for periodontal regeneration. |
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