Enhanced reconstruction of rat calvarial defects achieved by plasma-treated electrospun scaffolds and induced pluripotent stem cells

Tissue engineering with a combination of stem cells and nanofibrous scaffolds has attracted interest with regard to bone regeneration applications. In the present study, human induced pluripotent stem cells (iPSCs) were cultured on polymeric nanofibrous polyethersulfone (PES) with and without plasma treatment. The capacity of PES and plasma-treated PES (Plasma-PES) scaffolds to support the proliferation and osteogenic differentiation of iPSCs was investigated by MTT assay and for common osteogenic markers such as alkaline phosphatase activity, calcium mineral deposition and bone-related genes. Plasma-PES scaffolds with or without iPSCs were subsequently used to evaluate bone regeneration of critical-size defects in the rat by digital mammography, multislice spiral-computed tomography imaging and histological analysis. The results of in vitro analysis showed that plasma treatment significantly enhanced iPSC proliferation and osteogenesis. After 8 weeks of iPSC-loaded Plasma-PES implantation, no mortality or complication was observed in animals or at the site of surgery. Imaging analysis revealed more extensive bone reconstruction in rats receiving nanofibers compared with untreated control groups. Moreover, Plasma-PES seeded with iPSCs induced the highest regeneration of bone defects among all groups. These findings were confirmed by histological staining. Affective osseointegration was observed in implanted scaffolds. Thus, plasma-treated nanofibrous scaffolds are suitable tissue-engineered matrices for supporting the proliferation and osteogenic differentiation of iPSCs and might also be appropriate for the reconstruction of bone defects.     

Tissue transglutaminase regulates chondrogenesis in mesenchymal stem cells on collagen type XI matrices

Tissue transglutaminase (tTG) is a multifunctional enzyme with a plethora of potential applications in regenerative medicine and tissue bioengineering. In this study, we examined the role of tTG as a regulator of chondrogenesis in human mesenchymal stem cells (MSC) using nanofibrous scaffolds coated with collagen type XI. Transient treatment of collagen type XI films and 3D scaffolds with tTG results in enhanced attachment of MSC and supports rounded cell morphology compared to the untreated matrices or those incubated in the continuous presence of tTG. Accordingly, enhanced cell aggregation and augmented chondrogenic differentiation have been observed on the collagen type XI-coated poly-(L-lactide) nanofibrous scaffolds treated with tTG prior to cell seeding. These changes implicate that MSC chondrogenesis is enhanced by the tTG-mediated modifications of the collagen matrix. For example, exogenous tTG increases resistance to collagenolysis in collagen type XI matrices by catalyzing intermolecular cross-linking, detected by a shift in the denaturation temperature. In addition, tTG auto-crosslinks to collagen type XI as detected by western blot and immunofluorescent analysis. This study identifies tTG as a novel regulator of MSC chondrogenesis further contributing to the expanding use of these cells in cartilage bioengineering.     

The restoration of full-thickness cartilage defects with mesenchymal stem cells (MSCs) loaded and cross-linked bilayer collagen scaffolds on rabbit model

Cartilage has a limited self-repair capability and the repair of large cartilage defects remains a challenge in clinic. This study aimed to investigate the effect of mesenchymal stem cells (MSCs) loaded three-dimensional bilayer collagen scaffold for cartilage repair. Cross-linked three-dimensional bilayer collagen scaffolds seeded with or without MSCs were implanted into large cartilage defects (4 mm in diameter; 3 mm in depth) in rabbit knees. The untreated cartilage defects served as control. The tissue response was evaluated at 6 and 12 weeks after implantation by general histology and semi-quantitative histological grading systems. In addition, the repaired tissues were evaluated by mechanical test at 12 weeks after implantation. The MSCs-loaded collagen scaffold group showed the most hyaline cartilage, highest histological scores and compressive modulus. Moreover, it showed a good integration with the subchondral bone and adjacent cartilage. The structure of the novel bilayer collagen scaffolds provided architectural support for the differentiation of MSCs and demonstrated successful induction of in vivo chondrogenesis. These findings suggested that MSCs-loaded bilayer collagen scaffold could be an appealing candidate to be used for cartilage regeneration.     

Rapid human-derived iPSC osteogenesis combined with three-dimensionally printed Ti6Al4V scaffolds for the repair of bone defects

Human-induced pluripotent stem cells (iPSCs) are an alternative source of mesenchymal stem cells used for bone regeneration. However, the current osteogenically induced methods for iPSCs are slow and complex. We have used retinoic acid (RA) to induce osteogenic iPSCs within 10 days and assess whether a rapid differentiation could improve the osteogenic potential of the three-dimensionally printed Ti6Al4V (3DTi) scaffolds. First, the osteogenic differentiation of iPSCs was induced with RA, and the osteogenic potential of iPSCs was evaluated using standard assays. In addition, a 5-mm mandibular bone defect was generated in rats and was repaired with 3DTi scaffolds that were seeded with iPSC-induced osteoblasts. The capacity of seeded scaffolds for the enhancement of bone regeneration in vivo was assessed. Finally, we tested the potential mechanisms of RA-dependent iPSC bone induction and its effect on the Wnt/β-catenin pathway. The results showed that iPSCs could form osteocytes within 10 days. Animal experiments confirmed that rapid osteo-induced iPSCs could enhance the bone regeneration and osteointegration capacity of the 3DTi scaffolds. Mechanistically, RA could activate the AKT/GSK3β/β-catenin pathway during the process of iPSCs osteogenesis. The rapid osteoinduction of iPSCs combined with 3DTi scaffolds is a safe, effective, and reproducible method for repairing mandibular bone defects.     

Incorporated-bFGF polycaprolactone/polyvinylidene fluoride nanocomposite scaffold promotes human induced pluripotent stem cells osteogenic differentiation

Bioactive scaffolds that can increase transplanted cell survival time at the defect site have a great promising potential to use clinically since tissue regeneration or secretions crucially depend on the transplanted cell survival. In this study embedded basic fibroblast growth factor (bFGF)-polycaprolactone-polyvinylidene fluoride (PCL-PVDF) hybrid was designed and fabricated by electrospinning as a bio-functional nanofibrous scaffold for bone tissue engineering. After morphological characterization of the PCL-PVDF (bFGF) scaffold, nanofibers biocompatibility was investigated by culturing of the human induced pluripotent stem cells (iPSCs). Then, the bone differentiation capacity of the iPSCs was evaluated when grown on the PCL-PVDF and PCL-PVDF (bFGF) scaffolds in comparison with culture plate as a control using evaluating of the common osteogenic markers. The viability assay displayed a significant increase in iPSCs survival rate when grown on the bFGF content scaffold. The highest alkaline phosphatase activity and mineralization were detected in the iPSCs while grown on the PCL-PVDF (bFGF) scaffolds. Obtained results from gene and protein expression were also demonstrated the higher osteoinductive property of the bFGF content scaffold compared with the scaffold without it. According to the results, the release of bFGF from PCL-PVDF nanofibers increased survival and proliferation rate of the iPSCs, which followed by an increase in its osteogenic differentiation potential. Taking together, PCL-PVDF (bFGF) nanofibrous scaffold demonstrated that can be noted as a promising candidate for treating the bone lesions by tissue engineering products.     

Improved osteogenic differentiation of human induced pluripotent stem cells cultured on polyvinylidene fluoride/collagen/platelet-rich plasma composite nanofibers

Blood transfusion or blood products, such as plasma, have a long history in improving health, but today, platelet-rich plasma (PRP) is used in various medical areas such as surgery, orthopedics, and rheumatology in many ways. Considering the high efficiency of tissue engineering in repairing bone defects, in this study, we investigated the combined effect of nanofibrous scaffolds in combination with PRP on the osteogenic differentiation potential of human induced pluripotent stem cells (iPSCs). Electrospinning was used for fabricating nanofibrous scaffolds by polyvinylidene fluoride/collagen (PVDF/col) with and without PRP. After scaffold characterization, the osteoinductivity of the fabricated scaffolds was studied by culturing human iPSCs under osteogenic medium. The results showed that PRP has a considerable positive effect on the biocompatibility of the PVDF/col nanofibrous scaffold when examined by protein adsorption, cell attachment, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. In addition, the results obtained from alkaline phosphatase activity and calcium content assays demonstrated that nanofibers have higher osteoinductivity while grown on PRP-incorporated PVDF/col nanofibers. These results were also confirmed while the osteogenic differentiation of the iPSCs was more investigated by evaluating the most important bone-related genes expression level. According to the results, it can be concluded that PVDF/col/PRP has much more osteoinductivity while compared with the PVDF/col, and it can be introduced as a promising bone bio-implant for use in bone tissue engineering applications.     

A growth factor delivery system for chondrogenic induction of infrapatellar fat pad-derived stem cells in fibrin hydrogels

Articular cartilage has a limited capacity for self-renewal and repair. Tissue engineering of cartilage in vitro has been proposed as a solution to this problem; however, this approach is costly and requires a significant amount of time to grow the graft. An alternative approach is to implant chondroprogenitor cells seeded within a growth factor delivery scaffold directly into the defect site to promote tissue regeneration. The objective of this study was to develop a biocompatible growth factor delivery system capable of promoting chondrogenesis of infrapatellar fat pad (IFP)-derived stem cells. Transforming growth factor beta-1 (TGF-β1) was loaded into gelatin microspheres and incorporated into fibrin hydrogels containing IFP-derived stem cells. The release of TGF-β1 was quantified using an enzyme-linked immunosorbent assay, whereas chondrogenesis was demonstrated histologically and by quantifying sulfated glycosaminoglycan production after 21 days of in vitro culture. TGF-β1 loaded into gelatin microspheres appeared to be as effective in promoting chondrogenesis of IFP-derived stem cells as adding TGF-β1 directly to the medium. The influence of different microsphere fabrication parameters and TGF-β1 loading concentrations was also investigated but appeared to only have a small effect on subsequent chondrogenesis. The development of such growth factor delivery systems in combination with IFP-derived stem cells represents a potential new strategy for cartilage defect repair.     

Platelet-rich plasma promotes the regeneration of cartilage engineered by mesenchymal stem cells and collagen hydrogel via the TGF-β/SMAD signaling pathway

The tissue engineering technique using mesenchymal stem cells (MSCs) and scaffolds is promising. Transforming growth factor-β1 (TGF-β1) is generally accepted as an chondrogenic agent, but immunorejection and unexpected side effects, such as tumorigenesis and heterogeneity, limit its clinical application. Autogenous platelet-rich plasma (PRP), marked by low immunogenicity, easy accessibility, and low-cost, may be favorable for cartilage regeneration. In our study, the effect of PRP on engineered cartilage constructed by MSCs and collagen hydrogel in vitro and in vivo was investigated and compared with TGF-β1. The results showed that PRP promoted cell proliferation and gene and protein expressions of chondrogenic markers via the TGF-β/SMAD signaling pathway. Meanwhile, it suppressed the expression of collagen type I, a marker of fibrocartilage. Furthermore, PRP accelerated cartilage regeneration on defects with engineered cartilage, advantageous over TGF-β1, as evaluated by histological analysis and immunohistochemical staining. Our work demonstrates that autogenous PRP may substitute TGF-β1 as a potent and reliable chondrogenic inducer for therapy of cartilage defect.     

Dynamic compression of rabbit adipose-derived stem cells transfected with insulin-like growth factor 1 in chitosan/gelatin scaffolds induces chondrogenesis and matrix biosynthesis

Articular cartilage is routinely subjected to mechanical forces and growth factors. Adipose-derived stem cells (ASCs) are multi-potent adult stem cells and capable of chondrogenesis. In the present study, we investigated the comparative and interactive effects of dynamic compression and insulin-like growth factor-I (IGF-I) on the chondrogenesis of rabbit ASCs in chitosan/gelatin scaffolds. Rabbit ASCs with or without a plasmid overexpressing of human IGF-1 were cultured in chitosan/gelatin scaffolds for 2 days, then subjected to cyclic compression with 5% strain and 1 Hz for 4 h per day for seven consecutive days. Dynamic compression induced chondrogenesis of rabbit ASCs by activating calcium signaling pathways and up-regulating the expression of Sox-9. Dynamic compression plus IGF-1 overexpression up-regulated expression of chondrocyte-specific extracellular matrix genes including type II collagen, Sox-9, and aggrecan with no effect on type X collagen expression. Furthermore, dynamic compression and IGF-1 expression promoted cellular proliferation and the deposition of proteoglycan and collagen. Intracellular calcium ion concentration and peak currents of Ca(2+) ion channels were consistent with chondrocytes. The tissue-engineered cartilage from this process had excellent mechanical properties. When applied together, the effects achieved by the two stimuli (dynamic compression and IGF-1) were greater than those achieved by either stimulus alone. Our results suggest that dynamic compression combined with IGF-1 overexpression might benefit articular cartilage tissue engineering in cartilage regeneration.     

Deletion of Alox5 gene decreases osteogenic differentiation but increases adipogenic differentiation of mouse induced pluripotent stem cells

Induced pluripotent stem cells (iPSCs) have great potential in bone tissue engineering to repair large bone defects. Before their clinical application, investigations are needed to discover the genes and osteoconductive scaffolds that influence their differentiation toward an osteogenic lineage. Alox5 plays controversial and complex roles in the regulation of bone and fat metabolism. To detect the effect of Alox5 on osteogenic and adipogenic differentiation of iPSCs, both Alox5 knockout mouse iPSCs (Alox5-KO-iPSCs) and wild-type mouse iPSCs (Wild-iPSCs) were developed. The mRNA levels of many osteogenic markers in Alox5-KO-iPSCs were significantly reduced, while many adipogenic markers were enhanced. Furthermore, when implanted in rat cranial critical-sized defects with collagen/chitosan/hydroxyapatite scaffolds (CCHS), Alox5-KO-iPSCs produced significantly less new bone than Wild-iPSCs and both cell-scaffold groups had no tumor formation. There was a significant difference in the expression of Cox2 during the osteogenic and adipogenic differentiation between the two kinds of iPSCs in vitro. In conclusion, firstly, Alox5 knockout reduced the osteogenic but increased the adipogenic differentiation potential of mouse iPSCs. These disorders might be related to the change of Cox2 expression. Secondly, combined with iPSCs, CCHS can serve as a potential substrate to repair critical-sized bony defects. However, more studies are required to confirm the mechanisms through which Alox5 affects the osteogenic and adipogenic abilities of iPSCs in vivo and the effect of Cox2 inhibition in this system.     

Rescue of proinflammatory cytokine-inhibited chondrogenesis by the antiarthritic effect of melatonin in synovium mesenchymal stem cells via suppression of reactive oxygen species and matrix metalloproteinases

Cartilage repair by mesenchymal stem cells (MSCs) often occurs in diseased joints in which the inflamed microenvironment impairs chondrogenic maturation and causes neocartilage degradation. In this environment, melatonin exerts an antioxidant effect by scavenging free radicals. This study aimed to investigate the anti-inflammatory and chondroprotective effects of melatonin on human MSCs in a proinflammatory cytokine-induced arthritic environment. MSCs were induced toward chondrogenesis in the presence of interleukin-1 β (IL-1β) or tumor necrosis factor α (TNF-α) with or without melatonin. Levels of intracellular reactive oxygen species (ROS), hydrogen peroxide, antioxidant enzymes, and cell viability were then assessed. Deposition of glycosaminoglycans and collagens was also determined by histological analysis. Gene expression of chondrogenic markers and matrix metalloproteinases (MMPs) was assessed by real-time polymerase chain reaction. In addition, the involvement of the melatonin receptor and superoxide dismutase (SOD) in chondrogenesis was investigated using pharmacologic inhibitors. The results showed that melatonin significantly reduced ROS accumulation and increased SOD expression. Both IL-1β and TNF-α had an inhibitory effect on the chondrogenesis of MSCs, but melatonin successfully restored the low expression of cartilage matrix and chondrogenic genes. Melatonin prevented cartilage degradation by downregulating MMPs. The addition of luzindole and SOD inhibitors abrogated the protective effect of melatonin associated with increased levels of ROS and MMPs. These results demonstrated that proinflammatory cytokines impair the chondrogenesis of MSCs, which was rescued by melatonin treatment. This chondroprotective effect was potentially correlated to decreased ROS, preserved SOD, and suppressed levels of MMPs. Thus, melatonin provides a new strategy for promoting cell-based cartilage regeneration in diseased or injured joints.     

Composite hyaluronate-type I collagen-fibrin scaffold in the therapy of osteochondral defects in miniature pigs

The potential of novel scaffold containing sodium hyaluronate, type I collagen, and fibrin was investigated in the regeneration of osteochondral defects in miniature pigs. Both autologous chondrocyte-seeded scaffolds and non-seeded scaffolds were implanted into two defects located in the non-weight-bearing zone of the femoral trochlea (defect A was located more distally and medially, defect B was located more proximally and laterally). Control defects were left untreated. Twelve weeks after the operation, the knees were evaluated in vivo using MRI. Six months after the implantation, the defects were analyzed using MRI, histological, and immunohistochemical analysis. In the A defects of chondrocyte-seeded scaffold group, hyaline cartilage and fibrocartilage was formed, containing type II collagen, acidic and neutral glycosaminoglycans while the non-seeded scaffold group was predominantly filled with fibrocartilage. Defects in the control group were predominantly filled with fibrous tissue. Histomorphometric analysis of photomicrographs revealed a significantly higher amount of hyaline cartilage in the cell-seeded scaffold group in A defects than in other groups. Both scaffold groups in A defects showed significantly less fibrous tissue than cell-seeded defects B and the control group. Both histological and MRI analysis proved that the novel composite scaffold has a potential to regenerate osteochondral defects within six months.     

Treatment of Osteochondral Defects in the Rabbit's Knee Joint by Implantation of Allogeneic Mesenchymal Stem Cells in Fibrin Clots

The treatment of osteochondral articular defects has been challenging physicians for many years. The better understanding of interactions of articular cartilage and subchondral bone in recent years led to increased attention to restoration of the entire osteochondral unit. In comparison to chondral lesions the regeneration of osteochondral defects is much more complex and a far greater surgical and therapeutic challenge. The damaged tissue does not only include the superficial cartilage layer but also the subchondral bone. For deep, osteochondral damage, as it occurs for example with osteochondrosis dissecans, the full thickness of the defect needs to be replaced to restore the joint surface ¹. Eligible therapeutic procedures have to consider these two different tissues with their different intrinsic healing potential ². In the last decades, several surgical treatment options have emerged and have already been clinically established ^3-6.Autologous or allogeneic osteochondral transplants consist of articular cartilage and subchondral bone and allow the replacement of the entire osteochondral unit. The defects are filled with cylindrical osteochondral grafts that aim to provide a congruent hyaline cartilage covered surface ^3,7,8. Disadvantages are the limited amount of available grafts, donor site morbidity (for autologous transplants) and the incongruence of the surface; thereby the application of this method is especially limited for large defects.New approaches in the field of tissue engineering opened up promising possibilities for regenerative osteochondral therapy. The implantation of autologous chondrocytes marked the first cell based biological approach for the treatment of full-thickness cartilage lesions and is now worldwide established with good clinical results even 10 to 20 years after implantation ^9,10. However, to date, this technique is not suitable for the treatment of all types of lesions such as deep defects involving the subchondral bone ¹¹.The sandwich-technique combines bone grafting with current approaches in Tissue Engineering ^5,6. This combination seems to be able to overcome the limitations seen in osteochondral grafts alone. After autologous bone grafting to the subchondral defect area, a membrane seeded with autologous chondrocytes is sutured above and facilitates to match the topology of the graft with the injured site. Of course, the previous bone reconstruction needs additional surgical time and often even an additional surgery. Moreover, to date, long-term data is missing ¹².Tissue Engineering without additional bone grafting aims to restore the complex structure and properties of native articular cartilage by chondrogenic and osteogenic potential of the transplanted cells. However, again, it is usually only the cartilage tissue that is more or less regenerated. Additional osteochondral damage needs a specific further treatment. In order to achieve a regeneration of the multilayered structure of osteochondral defects, three-dimensional tissue engineered products seeded with autologous/allogeneic cells might provide a good regeneration capacity ¹¹.Beside autologous chondrocytes, mesenchymal stem cells (MSC) seem to be an attractive alternative for the development of a full-thickness cartilage tissue. In numerous preclinical in vitro and in vivo studies, mesenchymal stem cells have displayed excellent tissue regeneration potential ^13,14. The important advantage of mesenchymal stem cells especially for the treatment of osteochondral defects is that they have the capacity to differentiate in osteocytes as well as chondrocytes. Therefore, they potentially allow a multilayered regeneration of the defect.In recent years, several scaffolds with osteochondral regenerative potential have therefore been developed and evaluated with promising preliminary results ^1,15-18. Furthermore, fibrin glue as a cell carrier became one of the preferred techniques in experimental cartilage repair and has already successfully been used in several animal studies ^19-21 and even first human trials ²².The following protocol will demonstrate an experimental technique for isolating mesenchymal stem cells from a rabbit''s bone marrow, for subsequent proliferation in cell culture and for preparing a standardized in vitro-model for fibrin-cell-clots. Finally, a technique for the implantation of pre-established fibrin-cell-clots into artificial osteochondral defects of the rabbit''s knee joint will be described.     

Cartilage resurfacing potential of PLGA scaffolds loaded with autologous cells from cartilage,fat, and bone marrow in an ovine model of osteochondral focal defect

Current developments in tissue engineering strategies for articular cartilage regeneration focus on the design of supportive three-dimensional scaffolds and their use in combination with cells from different sources. The challenge of translating initial successes in small laboratory animals into the clinics involves pilot studies in large animal models, where safety and efficacy should be investigated during prolonged follow-up periods. Here we present, in a single study, the long-term (up to 1 year) effect of biocompatible porous scaffolds non-seeded and seeded with fresh ex vivo expanded autologous progenitor cells that were derived from three different cell sources [cartilage, fat and bone marrow (BM)] in order to evaluate their advantages as cartilage resurfacing agents. An ovine model of critical size osteochondral focal defect was used and the test items were implanted arthroscopically into the knees. Evidence of regeneration of hyaline quality tissue was observed at 6 and 12 months post-treatment with variable success depending on the cell source. Cartilage and BM-derived mesenchymal stromal cells (MSC), but not those derived from fat, resulted in the best quality of new cartilage, as judged qualitatively by magnetic resonance imaging and macroscopic assessment, and by histological quantitative scores. Given the limitations in sourcing cartilage tissue and the risk of donor site morbidity, BM emerges as a preferential source of MSC for novel cartilage resurfacing therapies of osteochondral defects using copolymeric poly-d,l-lactide-co-glycolide scaffolds.     

骨碎补结合组织工程软骨促进软骨再生的实验研究

目的:评估骨碎补结合组织工程软骨治疗对实验兔软骨缺陷模型软骨再生的疗效。方法:将h IGF-1基因转染MSCs,并与脱细胞真皮基质(ADM)构建组织工程软骨。24只新西兰白兔随机分为A、B、C、D四组,A、C组进行自体软骨移植,B、D组进行改建的细胞-ADM移植。C、D组用40%骨碎补汤喂养4周,150 m L/d。第12周处死实验动物,分离缺损关节软骨部位,蜡块包埋染色,通过总体形态评价软骨再生组织。采用组织学评分评估再生软骨质量。采用甲苯胺蓝染色评价缺损部位产生软骨糖胺聚糖的情况。结果:与B组比较,C组和D组的新生软骨覆盖度、新骨髓的颜色、缺损边缘和表面粗糙度均显著提高(P0.05);再生软骨的组织学评分软骨表面评分显著改善(P0.05)。C组与D组具有比其他组更好的基质、细胞分布和表面指数。并且有较厚的透明样软骨组织,具有正常的糖胺聚糖产生。表明该治疗方法可以通过再生透明样软骨且没有不良事件来减少软骨缺陷。结论:工程软骨结合骨碎补治疗可显著改善兔膝关节软骨缺损修复的质量,为临床治疗软骨病变提供重要理论依据。     

The effect of autologous bone marrow stromal cells differentiated on scaffolds for canine tibial bone reconstruction

Bone marrow contains mesenchymal stem cells that form many tissues. Various scaffolds are available for bone reconstruction by tissue engineering. Osteoblastic differentiated bone marrow stromal cells (BMSC) promote osteogenesis on scaffolds and stimulate bone regeneration. We investigated the use of cultured autologous BMSC on different scaffolds for healing defects in tibias of adult male canines. BMSC were isolated from canine humerus bone marrow, differentiated into osteoblasts in culture and loaded onto porous ceramic scaffolds including hydroxyapatite 1, hydroxyapatite gel and calcium phosphate. Osteoblast differentiation was verified by osteonectine and osteocalcine immunocytochemistry. The scaffolds with stromal cells were implanted in the tibial defect. Scaffolds without stromal cells were used as controls. Sections from the defects were processed for histological, ultrastructural, immunohistochemical and histomorphometric analyses to analyze the healing of the defects. BMSC were spread, allowed to proliferate and differentiate to osteoblasts as shown by alizarin red histochemistry, and osteocalcine and osteonectine immunostaining. Scanning electron microscopy showed that BMSC on the scaffolds were more active and adhesive to the calcium phosphate scaffold compared to the others. Macroscopic bone formation was observed in all groups, but scaffolds with stromal cells produced significantly better results. Bone healing occurred earlier and faster with stromal cells on the calcium phosphate scaffold and produced more callus compared to other scaffolds. Tissue healing and osteoblastic marker expression also were better with stromal cells on the scaffolds. Increased trabecula formation, cell density and decreased fibrosis were observed in the calcium phosphate scaffold with stromal cells. Autologous cultured stromal cells on the scaffolds were useful for healing of canine tibial bone defects. The calcium phosphate scaffold was the best for both cell differentiation in vitro and bone regeneration in vivo. It may be possible to improve healing of bone defects in humans using stem cells from bone marrow.     

Porous Silk Scaffolds for Delivery of Growth Factors and Stem Cells to Enhance Bone Regeneration

Stem cell-based tissue engineering shows promise for bone regeneration and requires artificial microenvironments to enhance the survival, proliferation and differentiation of the seeded cells. Silk fibroin, as a natural protein polymer, has unique properties for tissue regeneration. The present study aimed to evaluate the influence of porous silk scaffolds on rat bone marrow stem cells (BMSCs) by lenti-GFP tracking both in vitro and in vivo in cranial bone defects. The number of cells seeded within silk scaffolds in rat cranial bone defects increased from 2 days to 2 weeks after implantation, followed by a decrease at eight weeks. Importantly, the implanted cells survived for 8 weeks in vivo and some of the cells might differentiate into endothelial cells and osteoblasts induced by the presence of VEGF and BMP-2 in the scaffolds to promote angiogenesis and osteogenesis. The results demonstrate that porous silk scaffolds provide a suitable niche to maintain long survival and function of the implanted cells for bone regeneration.     

Evaluating the bone regeneration in calvarial defect using osteoblasts differentiated from adipose-derived mesenchymal stem cells on three different scaffolds: an animal study

The aim of this study was to investigate the effect of three different scaffolds on the viability and differentiation of adipose-derived mesenchymal stem cells (ADMSCs) to osteoblast for bone regeneration of calvarial defect in rabbit model. Adipose was harvested from the nape of 12 rabbits by direct surgery or hollow-tip cannula. Two standardized circular calvarial defects (case and control), 8 mm in diameter each, were created in all the animals. The animals were divided into 3 different groups. In group 1 (G1), the defect was filled with polyamide + ADMSC. In group 2, poly lactic-co-glycolic acid + ADMSC was used. In group 3, decellularized amniotic membrane + ADMSC was applied. In the control defect, the non-seeded scaffolds were applied for filling the defect. Decellularized pericardial scaffolds were used as a membrane on the scaffolds. The animals were euthanized 2, 4, and 8 weeks of operation and new bone formation was assessed by different analyses. Immunohistochemical (IHC) staining with osteopontin and osteocalcin antibodies was also performed. After 2 weeks of wound healing, minimal bone regeneration was detected in all groups. Almost complete defect closure was observed in all experimental groups after 8 weeks of operation, with the greatest defect closure in the animals treated with polyamide scaffolds as compared to biopsies obtained from control defects and other experimental groups. The maximal tensile load was higher in G1, 4 and 8 weeks postoperatively, suggesting the usefulness of polyamide + ADMSC for bone regeneration in calvarial defects. Results of the IHC staining demonstrated a significant difference between seeded and non-seeded scaffold in both short- and long-term follow-ups (P < 0.05). In addition, a significant difference was observed in enhancement of IHC staining of both markers in polyamide group (seeded or non-seeded) 4 and 8 weeks postoperatively in comparison with other scaffolds. It was concluded that bone regeneration in critical calvarial defect was more successful in seeded polyamide.