| Periodontal regeneration involves the restoration of at least three unique tissues: cementum, periodontal ligament(PDL) and alveolar bone tissue. Here, we first isolated human PDL stem cells(PDLSCs) and jaw bone mesenchymal stem cells(JBMSCs). These cells were then induced to form cell sheets using an ascorbic acid-rich approach, and the cell sheet properties, including morphology, thickness and gene expression profile, were compared. Afterwards, platelet-rich fibrin(PRF) derived from human venous blood was fabricated into bioabsorbable fibrin scaffolds containing various growth factors. Finally, a cell-material construct based on PDLSC sheets, PRF scaffolds and JBMSC sheets, as well as human treated dentin matrix(TDM) and hydroxyapatite(HA)/tricalcium phosphate(TCP), was used to regenerate the periodontal complex in a nude mouse model. In this model, PDLSC sheet/PRF/JBMSC sheet composites were placed in a simulated periodontal space consisting of human TDM and HA/TCP frameworks. Eight weeks after implantation, the PDLSC sheets tended to develop into PDL- and cementum-like tissues, whereas the JBMSC sheets tended to produce predominantly bone-like tissues. Furthermore, the PDLSC sheet/PRF/JBMSC sheet composites generated periodontal complex-like structures containing PDL-, cementum- and bone-like tissues. This cell transplantation method may provide an effective approach for future periodontal complex regeneration in clinical practice.Part I:Isolation and characterization of human periodontal ligament stem cells(PDLSCs) and jaw bone mesenchymal stem cells(JBMSCs)Objective:To isolate human PDLSCs and JBMSCs and compared their differentiation properties.Methods:The PDL was isolated from extracted second premolars from orthodontic patients(18-23 years of age, n=3).The PDL tissues in the middle portion of the root surfaces were carefully scraped, washed repeatedly with PBS, and then cut into small blocks(approximately 1 mm3). The tissue blocks were digested in 5 m L of α-minimum essential medium(α-MEM; Hyclone, MA, USA) containing 1% collagenase type I and 1% dispase(both from Sigma-Aldrich, St. Louis, MO, USA) for 40 min at 37°C. The digested tissues were transferred to 3.5-cm-diameter culture dishes. For the isolation of JBMSCs, fresh cancellous bone fragments and blood were obtained from three orthognathic patients(20-26 years of age) via mandibular angle resection. The fragments were rapidly transferred to a clean bench. A syringe device was used to pour α-MEM repeatedly and gently over the fragments until the fragments displayed a white appearance. Then, the mixed cell types in medium were centrifuged, resuspended, and seeded in a six-well plate(Corning) containing complete medium. The colony-forming unit-fibroblast(CFU-F) assays were employed to determine the colony-forming abilities of the stem cells. The cell counting kit-8 assay was used to quantitatively evaluate the number of proliferating cells during a 7-day culture period in complete medium. Flow cytometric analysis was adopted to determine the expression of cell surface markers of PDLSCs and JBMSCs(P3) as described previously To assess the osteogenic ability of each cell type, the osteoinduction medium was replaced at 3-day intervals. Following 4 weeks of osteogenic induction, the cells were fixed in 4% paraformaldehyde and then stained with Alizarin Red S staining solution(Sigma–Aldrich) for 3 min at room temperature. Afterwards, the dishes were w observed under a microscope. To assess adipogenesis, the adipogenic medium(complete medium supplemented with 100 n M dexamethasone, 10μg/m L insulin, 0.5 m M 3-isobutyl-1-methylxanthine, and 50 m M indomethacin) was replaced every three days. After two weeks, the cells were fixed in 4% paraformaldehyde and stained with Oil Red O(Sigma–Aldrich) staining solution for 15 min. Afterwards, the dishes were washed twice with PBS and observed under a microscope in PBS. For subcutaneous implantations of in vitro-expanded PDLSCs(P4) and JBMSCs(P4), 2.0×106 culture-expanded cells were suspended in 0.5 m L of complete medium, mixed with 40 mg of HA/TCP and incubated for 90 min at 37°C. The cell/carrier mixtures were immediately implanted into the dorsum of nude mice. At eight weeks post-implantation, all nude mice were sacrificed. Then, 3-D reconstruction images of the grafts were obtained using micro-CT, and the equivalent bone density of each sample was calculated and used for statistical analysis. Then, the harvested samples were fixed with 4% paraformaldehyde for 48 h at 4°C, demineralized with 10% EDTA(p H 6.9) at room temperature for 2 weeks, and embedded in paraffin. Afterwards, the paraffinized sections were stained with haematoxylin and eosin(H&E) for observation of the regenerated tissue. Real-time PCR was employed to determine the differences in gene expression between PDLSCs(P2) and JBMSCs(P2).Results: After 5-7 days in primary culture, PDLSCs and JBMSCs successfully proliferated, forming cells displaying spindle-shaped morphologies and single-cell clones of varying sizes. Both putative stem cells demonstrated colony formation ability, although the number of CFU-Fs formed by PDLSCs was higher than that formed by JBMSCs(P<0.01). Similarly, the slope of the cell growth curve following a 7-day incubation for PDLSCs was generally much steeper than that for JBMSCs, and the cell number in each group peaked on day 6; significant differences in cell proliferation were detected on days 4,5, 6 and 7 between these two putative stem cell types(P<0.01). The immunophenotype of PDLSCs and JBMSCs was determined via flow cytometry. In general, both putative stem cell types were negative for the haematopoietic markers CD34 and CD45 and were positive for the mesenchymal-associated marker CD29. In addition, PDLSCs and JBMSCs expressed STRO-1 and CD146, both of which are early markers of MSCs. Furthermore, the rate of CD146 positivity was higher among PDLSCs(P<0.05), although no significant difference in the frequency of expression of the other positive or negative markers was observed between these two cell types(P>0.05). PDLSCs and JBMSCs(P3) were induced in osteogenic or adipogenic media for several weeks to evaluate their multipotent differentiation potential. In terms of adipogenic differentiation, microscopic Oil Red O-positive lipid droplets were observed in both PDLSCs and JBMSCs after 2 weeks of induction. However, the JBMSCs tended to develop more and larger lipid droplets in the cells, and quantitative analysis showed that the OD was higher in JBMSCs than in PDLSCs(P<0.05). In terms of osteogenic differentiation, both PDLSCs and JBMSCs formed alizarin red-positive mineralized nodules after 4 weeks of incubation. However, JBMSCs appeared to accumulate more calcium deposits, and based on statistical analysis, the quantity of mineralized nodules was greater in JBMSCs than in PDLSCs(P<0.05). These results indicated that JBMSCs exhibit higher osteogenic potential than PDLSCs. To validate the osteogenic differentiation capacity of PDLSCs and JBMSCs in vivo, at eight weeks post-implantation, 3-D reconstruction images of the grafts were obtained using micro-CT scanning data. These images indicated that the JBMSC sheet/HA/TCP group displayed a higher density(more red areas) than the PDLSC sheet/HA/TCP group(more green areas). Quantitative analysis revealed that the equivalent bone density of the JBMSC sheet/HA/TCP group was higher than that of the PDLSC sheet/HA/TCP group(P<0.05). H&E staining of the sections revealed that the PDLSC sheet/HA/TCP group contained many collagenous fibers similar to those found in the PDL and few small bone-like structures; conversely, the JBMSC sheet/HA/TCP group contained many bone-like structures displaying various appearances and sizes but no evident collagenous fibers similar to those found in the PDL. The expression levels of eleven genes related to cellular stemness, adhesion, differentiation, and migration were analysed via real-time PCR. The results indicated that the gene expression profiles of PDLSCs and JBMSCs(P2) were very different. In general, compared to PDLSCs(P2) JBMSCs(P2) exhibited higher expression of stemness-related genes(nanog), adhesion-related genes(fibronectin and laminin), and calcification-related genes(col-I, col-III, periostin, bsp, opn and runx2), except for cemp-1, to varying degrees. Conclusion: We isolated two specific phenotypic stem cell types, PDLSCs and JBMSCs, for use as seeded cells and compared the differentiation tendencies of these cell types in vitro and in vivo. The results indicated that PDLSCs tend to produce PDL-/cementum-like tissues but that JBMSCs tend to generate bone-like tissues.Part II:Formation and characterization of PDLSC sheets and JBMSC sheetsObjective:To fabricate PDLSC and JBMSC sheets using an ascorbic acid-rich approach to more effectively load seeded cells. Furthermore, we investigated the characteristics of these composites in terms of morphology, thickness and gene expression profiles. Methods:PDLSCs(P3) and JBMSCs(P3) were separately seeded and cultured in sheet-inducing medium, consisting of complete medium supplemented with 50 μg/ml ascorbic acid; this medium was replaced every 3 days. After 14 days of incubation, the formed cell sheets were observed under an inverted microscope and then detached using ophthalmic forceps. For H&E staining, the detached cell sheets were fixed, embedded and sliced into 5-μm sections. Afterwards, the stained sections were observed photographically. For SEM observation, the detached cell sheets were fixed in 70% ethanol at 4°Cfor 30 min, dehydrated using a graded ethanol series, and dried. The surface and lateral section topographies of the cell sheets were observed via SEM(Hitachi S-4800; EIKO Engineering, Tokyo, Japan), and the sheet thickness was quantitatively measured from the photographs of the sections using Photoshop 7.0.For immunohistochemical staining, paraffinized sections(5 micrometers) of PDLSC or JBMSC sheets were deparaffinized, blocked, and incubated for one hour in the following primary antibodies:(1) anti-fibronectin(1:200),(2) anti-COLI(1:200),(3) anti-alkaline phosphatase(ALP; 1:200), and(4) anti-CEMP-1(1:200, all from Santa Cruz Biotechnologies, Dallas, TX, USA). Subsequently, the sections were incubated for 45 min in biotinylated secondary antibodies(1:1000) purchased from ZSGB BIO(Peking, China). Each experiment was repeated in triplicate. For real-time PCR analysis, total RNA was isolated from PDLSCs(P2), JBMSCs(P2), PDLSC sheets, and JBMSC sheets using TRIzol reagent. The c DNA synthesis and RT-PCR procedures were performed sequentially as described above. Finally, the relative expression levels of the target genes for each group were normalized to the expression level of the GAPDH gene. For Western blot analysis, PDLSC and JBMSC sheets were lysed in lysis buffer(Sigma–Aldrich) supplemented with 1 m M PMSF(Roche, Basel, Switzerland). Then, bicinchoninic acid(BCA) protein assay kit(Beyotime) was adopted to measure the total protein concentrations. Next, 40 μg of cell lysates were added to each lane of 10% SDS-PAGE gels, separated based on molecular weight, and electrotransferred to polyvinylidene fluoride(PVDF) membranes(Millipore, Billerica, MA, USA). After blocking with 5% non-fat milk for 2 h, the membranes were incubated in specific primary antibodies against COLI, COL III, OPN, OCN, CEMP-1, and CAP overnight at 4°C. Afterwards, membranes were incubated in a horseradish peroxidase(HRP)-conjugated anti-rabbit or anti-mouse secondary antibody(Co Win Biotech Co., Ltd., Beijing, China) for 1 hour at room temperature. The membranes were developed using the Western Light Chemiluminescent Detection System(Peiqing, Shanghai, China). All assays were repeated in triplicate.Results: Cell sheets were formed after a 14-day induction period. The cells became confluent and formed film-like cell sheets that could be completely detached from the edges of the dishes. Specifically, the JBMSC sheets appeared softer and thinner than the PDLSC sheets based on gross observation. We observed that the PDLSC and JBMSC sheets were arranged in a unidirectional or whirlpool-like pattern under an inverted microscope. Based on surface observation via SEM, both cell sheets contained many layers of cells that were closely arranged in an orderly pattern. However, based on observation of lateral sections via SEM and H&E staining, PDLSC sheets contained multiple layers of cells(3-7 layers), secreted a rich extracellular matrix(ECM) and tended to form a tight network of collagen fibres that retained tight junctions. Comparatively, the JBMSC sheets contained fewer layers of cells(2-4 layers) and secreted less extracellular matrix(ECM) than the PDLSC sheets. Based on quantitative analysis, the thickness of the PDLSC sheets was significantly greater than that of the JBMSC sheets(P<0.05). The alterations in gene expression from cells(P2) to cell sheets were determined via q RT-PCR. In general, the relative m RNA expression profiles of PDLSCs and JBMSCs were very different after forming cell sheets, and the expression of nearly every gene that we evaluated displayed an increasing trend after cell sheet formation. Compared to PDLSCs, PDLSC sheets exhibited high expression of periodontal tissue-specific genes(col-I, col-III, and periostin), calcification-related genes(runx2, bsp, opn, and ocn), and a cementum protein 1 gene(cemp-1), as well as slightly increased expression of adhesion-related genes(fibronectin and laminin)(Fig. 3B). Compared to JBMSCs, JBMSC sheets displayed high expression of calcification-related genes(runx2, bsp, opn, and ocn) and periodontal tissue-specific genes(col-I and col-III), as well as slightly increased expression of adhesion-related genes(fibronectin and laminin). We compared the relative gene expression profiles of PDLSC sheets and JBMSC sheets, and the results indicated that compared to PDLSC sheets JBMSC sheets exhibited increased expression of calcification-related genes(opn and ocn), adhesion-related genes(fibronectin and laminin), and periodontal tissue-specific genes(col-III and periostin), similar expression of col-I and runx2 and no expression of cemp-1. Furthermore, we compared the relative protein expression profiles of PDLSC sheets and JBMSC sheets via Western blot(Fig. 3E), and these results showed that JBMSC sheets exhibited higher expression of calcification-related proteins(COLIII, OPN, and OCN) and comparable expression of COLI and a cementum tissue-specific protein(CAP). Immunohistochemical staining indicated that both PDLSC and JBMSC sheets exhibited strong positive staining for COL1 and fibronectin, which are primarily found in ECM and which play important roles in maintaining biological functions. The expression of ALP, an early marker of osteoblast differentiation, was both observed a little positive in JBMSC sheets and in PDLSC sheets.Conclusion: The PDLSC sheets contained multiple(3-7) layers of cells, secreted rich ECM and tended to form a tight network of collagen fibres that retained tight junctions; alternatively, JBMSC sheets contained few(2-4) layers of cells and secreted a limited amount of ECM. PDLSCs and JBMSCs exhibited very different m RNA expression profiles after forming cell sheets, and the expression of nearly every gene that we evaluated displayed an increasing trend after cell sheet formation. JBMSC sheets exhibited higher expression of calcification-related genes(opn and ocn), adhesion-related genes(fibronectin and laminin) and periodontal tissue-specific genes(col-III and periostin) but lower expression of cemp-1 than PDLSC sheets. Moreover, we found similar results at the protein expression level between these two types of stem cell sheets based on Western blot assays. These results indicated that the differential gene expression profiles between PDLSCs and JBMSCs continue from primary cells to cell sheets and that these differences influence the structure and the function of newly formed tissues in vivo.Part III:In Vitro Studies on Human Periodontal Ligament Stem Cell Sheets Enhanced by Enamel Matrix DerivativeObjective : The present study investigated the effects of lyophilized EMD on the extracorporeal induction process and characteristics of PDL cell sheets for the exploration of more effective stem-cell therapy.Methods:Lactate dehydrogenase(LDH) activity was used as an index of cytotoxicity in the culture medium with α-MEM/10% FBS(control group) and α-MEM/10% FBS plus EMD(Enamel Matrix Derivative, Straumann, Malmo, Sweden) at concentrations of 25, 50 and 100 μg/m L.A cell counting kit-8 assay was used to quantitatively evaluate the number of proliferating cells. Cell sheets were cultured using regular sheet-inducing medium(complete medium supplemented with 50 μg/m L vitamin C; control group) or EMD-enhanced sheet-inducing medium(complete medium with 50 μg/m L vitamin C and 100 μg/m EMD; experimental groups) for 14 days. Cell sheets were observed by hematoxylin & eosin(H&E) staining, scanning electron microscopy(SEM), and immunohistochemical staining with the following primary antibodies:(1) anti-collagen type I(COL I) with a 1:200 dilution,(2) anti-alkaline phosphatase(OCN) with a 1:200 dilution, and(3) anti-cementum attachment protein(CAP) with a 1:200 dilution, all of which were obtained from Santa Cruz Biotechnology(Dallas, USA).. Real-time PCR was employed to assess the differences in gene expression. The following genes were monitored: collagen type Iα1(COL I), periostin(PERIOSTIN), runt-related transcription factor 2(RUNX2), osteopontin(OPN), osteocalcin(OCN), cementum attachment protein(CAP), and GAPDH. The relative expression of target genes was normalized to the expression of GAPDH. Cell sheets were cultured using regular sheet-inducing medium or EMD-enhanced sheet-inducing medium. At 14 days, the osteoinductive medium and osteoinductive medium supplemented with 100 μg/m EMD were switched and then refreshed at 3-day intervals. At 21 days(after 7 days of osteogenic induction), the cell sheets were fixed in 4% paraformaldehyde, and incubated with 2 m L of staining fluid(a mixture of 10 m L buffer, 33 m L 5-bromo-4-chloro-3-indolylphosphate(BCIP) and 66 m L nitroblue tetrazolium chloride(NBT), Beyotime) for 30 min at room temperature in the dark, and then washed with PBS. ALP activity was expressed as the integrated optical density(IOD) of the blue stain of the cytoplasm in representative images. The IOD was analyzed using Image-Pro plus 6.0 software(Media Cybernetics, Silver Spring, MD, USA). At 28 days(after 14 days of osteogenic induction), the cell sheets were fixed in 4% paraformaldehyde at and then stained with Alizarin Red Staining Solution(Sigma–Aldrich). The dishes were then washed twice with PBS, dried at room temperature and observed under a microscope. Finally, the mineralization nodules were dissolved in 2% cetylpyridinium chloride, and the OD values were measured at 560 nm using a spectrophotometer for statistical analysis. After 14 days of osteogenic induction, the cell sheets were detached, and subjected to SEM(Hitachi S-4300; EIKO Engineering, Tokyo, Japan) observation. The amounts of mineralized nodules were counted in five random SEM images(5000 x) for each group, and the data were subjected to statistical analysis.Results: The results showed no significant differences in LDH activity in the culture media among the different groups. Different EMD concentrations displayed different effects on cell proliferation. EMD(0, 25, 50 and 100 μg/m L) had a dose-dependent effect on the proliferation of PDLSCs; the 50 and 100 μg/m L EMD groups showed the highest cell numbers on d3 and d5, based on the statistical analysis(P< 0.01). Cell sheets were formed after a 14-day induction. Specifically, EMD-enhanced cell sheets appeared thicker and more compact than the normal induced PDLSC sheets by gross observation. Through surface observation by inverted microscopy and SEM, both cell sheets presented more layers of cells, which were orderly and closely arranged. Protein aggregates were formed on the surface of EMD-enhanced cell sheets under microscope due to an acidic solution of EMD being added to the culture medium. Based on lateral observation by H&E staining and SEM, EMD-enhanced cell sheets demonstrated more layers of cells(3-7 layers), secreted rich extracellular matrix(ECM) and tended to form a tight network of collagen fibers that retained tight junctions, while normal induced PDLSC sheets presented fewer layers of cells(2-4 layers) and secreted less ECM. When quantitatively analyzed through statistical analysis using lateral images taken by SEM, the thickness of EMD-enhanced cell sheets was significantly increased compared with that of normal induced sheets(P< 0.05). Immunohistochemical staining indicated that both EMD-enhanced cell sheets and normal induced cell sheets presented highly positive staining for COL1, which is mainly found in ECM and plays important roles in maintaining biological functions. Slightly increased ALP(an early marker of osteoblast differentiation) and CAP(a cementum-specific protein) expression was observed in EMD-enhanced cell sheets compared than in normal induced cell sheets, indicating the osteoblast and cementoblast differentiation tendencies of these EMD-enhanced cell sheets in vitro. Gene expression in cells(P3) and sheets on days 3, 7, and 14 was measured by real-time PCR. In general, the EMD-enhanced group showed relatively higher expression of periodontal tissue-specific genes(COL I, PERIOSTIN), calcification-related genes(RUNX2, OPN, OCN) and a cementum tissue-specific gene(CAP), to varying degrees, than that of cells and sheets cultured with normal medium on days 3, 7, 14. Cell sheets were cultured using EMD-enhanced sheet-inducing medium or normal sheet-inducing medium for 14 days and then subjected to osteogenic differentiation medium in vitro for 7 and 14 days. On day 7 of osteogenic differentiation, the ALP activities of cell sheets were detected by staining and were expressed as the mean IOD of the images assessed by using Image-Pro plus 6.0 software. The results showed that in the presence of the EMD, the ALP activities were significantly increased compared with those of the control group(P< 0.05). On day 14 of osteogenic differentiation, both types of cell sheets exhibited the potential to undergo osteogenic differentiation. In terms of Alizarin Red S staining and SEM observation, all cell sheets formed mineralized nodules, but EMD-enhanced cell sheets appeared to accumulate more calcium deposits. Based on the quantity of mineralized nodules, EMD-enhanced cell sheets had a much higher osteogenic potential than did normal induced cell sheets(P< 0.01; P< 0.05).Conclusion: In this work, we investigated the effects of lyophilized EMD on the extracorporeal induction process and characteristics of PDLSC sheets to explore a potentially more effective stem-cell therapy. EMD-enhanced cell sheets could be induced by complete medium supplemented with 50 μg/m L vitamin C and 100 μg/m L EMD. The EMD-enhanced cell sheets appeared thicker and more compact than the normal PDLSC sheets, demonstrated more layers of cells(3-7 layers), secreted richer ECM, and induced relatively higher m RNA expression of periodontal tissue-specific genes(COL I, PERIOSTIN), calcification-related genes(RUNX2, OPN, OCN) and a cementum tissue-specific gene(CAP), to varying degrees. Based on immunohistochemical results, slightly increased ALP and CAP protein expression was also observed in EMD-enhanced cell sheets than in normal induced cell sheets. In terms of the osteogenic differentiation of the cell sheets in vitro, EMD-enhanced cell sheets showed a stronger osteogenic capability. This type of EMD-enhanced cell sheet may represent a potential choice of stem-cell therapy for PDL regeneration.Part IV:In Vitro Studies on Human Periodontal Ligament Stem Cell Sheets Enhanced by Enamel Matrix DerivativeObjective:We fabricated PRF into a growth factor-rich scaffold, and treated dentin matrix(TDM)15 and hydroxyapatite(HA)/tricalcium phosphate(TCP) frameworks6 were produced to simulate the interfaces of dentin and alveolar bone, respectively, in a nude mouse implantation model. Finally, PDLSC sheet/PRF/JBMSC sheet composites were loaded in the simulated periodontal space formed by the TDM and HA/TCP frameworks, and the entire transplants were implanted into nude mice for 8 weeks to test the hypothesis that this method induces periodontal complex regeneration. We hope that this cell transplantation method will provide an approach for regeneration of the periodontal complex structure.Methods:10 m L of blood was rapidly withdrawn from the median cubital vein using a sterilized 10-m L syringe, transferred to test tubes lacking anticoagulant, and immediately centrifuged for 12 min at 400 g. Afterwards, the upper platelet-poor plasma layer was discarded, and the fibrin clot was separated from the red blood corpuscle(RBC) layer at the bottom using scissors. An at least 1-mm area of the RBC layer was carefully retained due to the concentrations of leukocytes and platelets at the junction zone. Several sterile dry gauzes were employed to softly compress the fibrin clot between the palms to squeeze out the fluids in the clot. As such, a resistant PRF membrane was obtained and used for subsequent experiments. The levels of crucial growth factors contained in one complete PRF membrane, i.e. TGF-β1, PDGF-AB, VEGF, IGF-1 and EGF, were quantified using ELISA kits(R&D Systems, Shanghai, China) based on the manufacturer’s instructions. To simulate the dentin components used for PDL tissue attachment, human TDM was prepared as described previously15. The root surfaces of the collected teeth were carefully cleaned and scraped using a curette to remove periodontal tissue. Then, the crowns at the cemento-enamel junction were rapidly removed using a high-speed dental turbine handpiece, and pre-dentin and dental pulp tissues were simultaneously discarded mechanically. Afterwards, the TDM was sequentially treated with 17% EDTA(Sigma, USA) for 5 min, 10% EDTA for 5 min and 5% EDTA for 10 min. Then, the TDM was mechanically cleaned twice in double-distilled water using an ultrasonic cleaner. Human TDM samples were air-dried and irradiated with ultraviolet light overnight on a super-clean bench, transferred to a sterile PBS solution containing 100 U/ml penicillin(Hyclone, USA) and 100 mg/ml streptomycin(Hyclone, USA) for 24 h, washed in sterile deionized water for 10 min in an ultrasonic cleaner, and stored in α-MEM at 4°C. To simulate the mineralized microenvironment from the alveolar bone, we constructed HA/TCP frameworks such that their inner contour matched the outer conical contour of the TDM. Holes that were larger than the human TDM were drilled inside the HA/TCP frameworks to ensure that there was sufficient space(0.5-1 mm) between these materials for the periodontium to regenerate from cell sheets and PRF. Finally, the HA/TCP frameworks were irradiated with cobalt 60 and stored in α-MEM at 4°C. To characterize the ultrastructure of the PRF membrane, the obtained PRF membrane fabricated as described above was fixed in 2.5% glutaraldehyde at 4°C for 30 min, dehydrated using a graded ethanol series and freeze-dried. The surface topography was observed via SEM. To characterize the ultrastructure of the TDM and HA/TCP frameworks, the biomaterials were processed as described above, dried, sputtered, and observed via SEM. To evaluate the periodontal regeneration potential of PDLSC and JBMSC sheets in vivo, the cell sheets, TDM and HA/TCP frameworks were transplanted into the subcutaneous dorsa of nude mice. Briefly, after 2 weeks of culture in 10-cm dishes, mature cell sheets were carefully extracted. The treated dentin matrix was carefully wrapped with at least three layers of PDLSC sheets, JBMSC sheets or PRF membrane and then placed in the inactivated HA/TCP frameworks. The above biomaterials were randomly divided into three groups according to their composition: group 1: PDLSC sheets/PRF/PDLSC sheets(order from the TDM side to the HA/TCP side); group 2: JBMSC sheets/PRF/JBMSC sheets; and group 3: PDLSC sheets/PRF/JBMSC sheets. Then, these complexes were incubated in complete medium containing 50 μg/ml ascorbic acid at 37°C for 90 min to facilitate stable adhesion. Afterwards, male 6-week-old nude mice were obtained from the animal centre of the Fourth Military Medical University. The cell sheets and the biomaterial complexes(n=5) were implanted into the subcutaneous dorsa of immunodeficient mice under deep anaesthesia via intraperitoneal injection of 10% chloral hydrate; then, the incisions for material implantation were carefully sutured. After eight weeks, samples were acquired and fixed in 4% paraformaldehyde for 2 days at 4°C. Then, the specimens were embedded in plastic and sectioned(20-30-μm-thick sections) using a hard tissue-slicing method. The sections were stained with H&E or Masson’s trichrome(Sigma-Aldrich, St. Louis, MO, USA). For immunohistochemical staining, the plastic sections were blocked, followed by incubation for 1 h in an anti-human COL-I primary antibody(1:200, Santa Cruz Biotechnologies, Dallas, TX, USA). PBS was applied instead of the primary antibody for negative controls. Subsequently, the specimens were incubated for 45 min in biotinylated secondary antibodies(1:1000) purchased from ZSGB BIO(Peking, China). The experiments were repeated in triplicate. All histological observations were performed using a light microscope(BX50, Olympus Optical, Japan), and photographs were obtained(DP25, Olympus). The results were collected and analysed by an independent observer to reduce bias and errors. To assess the ratio of newly formed PDL-like tissues to newborn bone-like tissues in each group, at least 3 randomly selected fields from each specimen were examined using Image-Pro Plus 6.0 software.Results: PRF was employed as a biological scaffold material containing absorbable fibrin and various human-derived growth factors. PRF clots were easily produced after centrifugation and were transformed into resistant fibrin membranes by compressing the fluids out of the fibrin matrix. SEM observation indicated that the PRF microstructure included abundant fibrin fibres and a few fibrillae of smaller diameter, constituting a 3-dimensional network. The upper white region of the PRF exhibited a neat appearance containing no cells, whereas the lower red region of the PRF contained many platelets, some leukocytes and few red blood cells, all of which were embedded in the network. ELISAs were performed to analyse the sequential profile of released growth factors in one complete PRF membrane over 7 days. The results revealed that the PRF membrane released growth factors in a time-dependent manner throughout the experimental period. Human TDM was fabricated using a previously described method to simulate dentin, which was produced to dimensions of 1.0 cm in length. After treatment with ethylene diamine tetra-acetic acid(EDTA), the dentinal tubules demonstrated an orderly arrangement based on SEM observation of their surface topography. The HA/TCP framework, which was used to simulate alveolar bone, was fabricated in a tooth root-like conformation in which HA/TCP particles of varying size were loosely and irregularly arranged such that numerous pores were present between the HA/TCP particles, as demonstrated by SEM observation. At 8 weeks after subcutaneous implantation into nude mice, PDLSC and JBMSC sheets produced very different structures. Histological observation showed that in group 1(PDLSC sheets/PRF/PDLSC sheets), many identically oriented collagen fibres were formed. Within these fibres, very rich blood vessel formation was observed within the ligament, and a thin layer of cementum-like structure was detected outside the large segments of the TDM. However, no bone-like tissues were evident. In group 2(JBMSC sheets/PRF/JBMSC sheets), abundant massive and continuous bone-like tissues, as well as several newly formed vessels, were observed. However, fewer collagen fibres were detected in group 2 than in group 1. In group 3(PDLSC sheets/PRF/JBMSC sheets), an interesting cementum/PDL/bone complex-like structure was observed. Briefly, a thin layer of cementum-like tissues covered the TDM surface, and outside this layer, a dense layer of connective tissue with a favourable orientation was detected. Outside these PDL-like tissues, several layers of massive and continuous bone-like tissues were observed. Finally, the r... |
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