Bioactive membranes for bone regeneration applications: Effect of physical and biomolecular signals on mesenchymal stem cell behavior

This study focuses on the in vitro characterization of bioactive elastin-like recombinamer (ELR) membranes for bone regeneration applications. Four bioactive ELRs exhibiting epitopes designed to promote mesenchymal stem cell adhesion (RGDS), endothelial cell adhesion (REDV), mineralization (HAP), and both cell adhesion and mineralization (HAP-RGDS) were synthesized using standard recombinant protein techniques. The materials were then used to fabricate ELR membranes incorporating a variety of topographical micropatterns including channels, holes and posts. Primary rat mesenchymal stem cells (rMSCs) were cultured on the different membranes and the effects of biomolecular and physical signals on cell adhesion, morphology, proliferation, and differentiation were evaluated. All results were analyzed using a custom-made MATLAB program for high throughput image analysis. Effects on cell morphology were mostly dependent on surface topography, while cell proliferation and cell differentiation were largely dependent on the biomolecular signaling from the ELR membranes. In particular, osteogenic differentiation (evaluated by staining for the osteoblastic marker osterix) was significantly enhanced on cells cultured on HAP membranes. Remarkably, cells growing on membranes containing the HAP sequence in non-osteogenic differentiation media exhibited significant up-regulation of the osteogenic marker as early as day 5, while those growing on fibronectin-coated glass in osteogenic differentiation media did not. These results are part of our ongoing effort to develop an optimized molecularly designed periosteal graft.     

Composite membranes of poly(lactic acid) with zinc-added bioactive glass as a guiding matrix for osteogenic differentiation of bone marrow mesenchymal stem cells

This study aims to produce a degradable and bone-bioactive membrane for guiding bone regeneration by combining a degradable polymer, poly(lactic acid) (PLA), with a bioactive inorganic zinc-containing bioactive glass (ZnBG). The in?vitro osteogenic development of rat bone marrow mesenchymal stem cells (rBMSCs) upon different membrane substrates (pure PLA control, PLA-BG, and PLA-ZnBG) was investigated in terms of bone cell phenotype syntheses and mineralization. Results showed significantly stimulated production of alkaline phosphatase and osteocalcin at days 14 and 21 in the membranes containing BG and ZnBG, with more in the samples containing ZnBG. The addition of ZnBG in PLA allowed the rBMSCs to express a high level of bone sialoprotein as confirmed by immunostaining. Cellular mineralization of the secreted extracellular matrix showed a significantly higher Ca level on the BG- and ZnBG-added membrane than on the PLA, and the more so in the ZnBG-added one. Based on the in?vitro assessments using rBMSCs, the ZnBG-added PLA is considered to be of potential use in guiding active bone regeneration within the periodontal pocket.     

Enhanced osteogenesis and angiogenesis by PCL/chitosan/Sr-doped calcium phosphate electrospun nanocomposite membrane for guided bone regeneration

Abstract

Membranes play pivotal role in guided bone regeneration (GBR) technique for reconstruction alveolar bone. GBR membrane that is able to stimulate both osteogenic and angiogenic differentiation of cells may be more effective in clinic practice. Herein, we fabricated the Sr-doped calcium phosphate/polycaprolactone/chitosan (Sr-CaP/PCL/CS) nanohybrid fibrous membrane by incorporating 20?wt% bioactive Sr-CaP nanoparticles into PCL/CS matrix via one-step electrospinning method, in order to endow the membrane with stimulation of osteogenesis and angiogenesis. The physicochemical properties, mechanical properties, Sr²⁺ release behavior, and the membrane stimulate bone mesenchymal stem cell (BMSCs) differentiation were evaluated in comparison with PCL/CS and CaP/PCL/CS membranes. The SEM images revealed that the nanocomposite membrane mimicked the extracellular matrix structure. The release curve presented a 28-day long continuous release of Sr²⁺ and concentration which was certified in an optimal range for positive biological effects at each timepoint. The in vitro cell culture experiments certified that the Sr-CaP/PCL/CS membrane enjoyed excellent biocompatibility and remarkably promoted rat bone mesenchymal stem cell (BMSCs) adhesion and proliferation. In terms of osteogenic differentiation, BMSCs seeded on the Sr-CaP/PCL/CS membrane showed a higher ALP activity level and a better matrix mineralization. What’s more, the synergism of the Sr²⁺ and CaP from the Sr-CaP/PCL/CS membrane enhanced BMSCs angiogenic differentiation, herein resulting in the largest VEGF secretion amount. Consequently, the Sr-CaP/PCL/CS nanohybrid electrospun membrane has promising applications in GBR.     

A comparison of bone regeneration with human mesenchymal stem cells and muscle-derived stem cells and the critical role of BMP

Adult multipotent stem cells have been isolated from a variety of human tissues including human skeletal muscle, which represent an easily accessible source of stem cells. It has been shown that human skeletal muscle-derived stem cells (hMDSCs) are muscle-derived mesenchymal stem cells capable of multipotent differentiation. Although hMDSCs can undergo osteogenic differentiation and form bone when genetically modified to express BMP2; it is still unclear whether hMDSCs are as efficient as human bone marrow mesenchymal stem cells (hBMMSCs) for bone regeneration. The current study aimed to address this question by performing a parallel comparison between hMDSCs and hBMMSCs to evaluate their osteogenic and bone regeneration capacities. Our results demonstrated that hMDSCs and hBMMSCs had similar osteogenic-related gene expression profiles and had similar osteogenic differentiation capacities in vitro when transduced to express BMP2. Both the untransduced hMDSCs and hBMMSCs formed very negligible amounts of bone in the critical sized bone defect model when using a fibrin sealant scaffold; however, when genetically modified with lenti-BMP2, both populations successfully regenerated bone in the defect area. No significant differences were found in the newly formed bone volumes and bone defect coverage between the hMDSC and hBMMSC groups. Although both cell types formed mature bone tissue by 6 weeks post-implantation, the newly formed bone in the hMDSCs group underwent quicker remodelling than the hBMMSCs group. In conclusion, our results demonstrated that hMDSCs are as efficient as hBMMSCs in terms of their bone regeneration capacity; however, both cell types required genetic modification with BMP in order to regenerate bone in vivo.     

Bioactive cell-derived matrices combined with polymer mesh scaffold for osteogenesis and bone healing

Successful bone tissue engineering generally requires an osteoconductive scaffold that consists of extracellular matrix (ECM) to mimic the natural environment. In this study, we developed a PLGA/PLA-based mesh scaffold coated with cell-derived extracellular matrix (CDM) for the delivery of bone morphogenic protein (BMP-2), and assessed the capacity of this system to provide an osteogenic microenvironment. Decellularized ECM from human lung fibroblasts (hFDM) was coated onto the surface of the polymer mesh scaffolds, upon which heparin was then conjugated onto hFDM via EDC chemistry. BMP-2 was subsequently immobilized onto the mesh scaffolds via heparin, and released at a controlled rate. Human placenta-derived mesenchymal stem cells (hPMSCs) were cultured in such scaffolds and subjected to osteogenic differentiation for 28 days in vitro. The results showed that alkaline phosphatase (ALP) activity, mineralization, and osteogenic marker expression were significantly improved with hPMSCs cultured in the hFDM-coated mesh scaffolds compared to the control and fibronectin-coated ones. In addition, a mouse ectopic and rat calvarial bone defect model was used to examine the feasibility of current platform to induce osteogenesis as well as bone regeneration. All hFDM-coated mesh groups exhibited a significant increase of newly formed bone and in particular, hFDM-coated mesh scaffold loaded with a high dose of BMP-2 exhibited a nearly complete bone defect healing as confirmed via micro-CT and histological observation. This work proposes a great potency of using hFDM (biophysical) coupled with BMP-2 (biochemical) as a promising osteogenic microenvironment for bone tissue engineering applications.     

A functional polyester carrying free hydroxyl groups promotes the mineralization of osteoblast and human mesenchymal stem cell extracellular matrix

Functional groups can control biointerfaces and provide a simple way to make therapeutic materials. We recently reported the design and synthesis of poly(sebacoyl diglyceride) (PSeD) carrying a free hydroxyl group in its repeating unit. This paper examines the use of this polymer to promote biomineralization for application in bone tissue engineering. PSeD promoted more mineralization of extracellular matrix secreted by human mesenchymal stem cells and rat osteoblasts than poly(lactic-co-glycolic acid) (PLGA), which is currently widely used in bone tissue engineering. PSeD showed in vitro osteocompatibility and in vivo biocompatibility that matched or surpassed that of PLGA, as well as supported the attachment, proliferation and differentiation of rat osteoblasts and human mesenchymal stem cells. This demonstrates the potential of PSeD for use in bone regeneration.     

Coating of VEGF-releasing scaffolds with bioactive glass for angiogenesis and bone regeneration

Bioactive glasses are potentially useful as bone defect fillers, and vascular endothelial growth factor (VEGF) has demonstrated benefit in bone regeneration as well. We hypothesized that the specific combination of prolonged localized VEGF presentation from a matrix coated with a bioactive glass may enhance bone regeneration. To test this hypothesis, the capacity of VEGF-releasing polymeric scaffolds with a bioactive glass coating was examined in vitro and in vivo using a rat critical-sized defect model. In the presence of a bioactive glass coating, we did not detect pronounced differences in the differentiation of human mesenchymal stem cells in vitro. However, we observed significantly enhanced mitogenic stimulation of endothelial cells in the presence of the bioactive glass coating, with an additive effect with VEGF release. This trend was maintained in vivo, where coated VEGF-releasing scaffolds demonstrated significant improvements in blood vessel density at 2 weeks versus coated control scaffolds. At 12 weeks, bone mineral density was significantly increased in coated VEGF-releasing scaffolds versus coated controls, while only a slight increase in bone volume fraction was observed. The results of this study suggest that a bioactive glass coating on a polymeric substrate participates in bone healing through indirect processes which enhance angiogenesis and bone maturation and not directly on osteoprogenitor differentiation and bone formation. The mass of bioactive glass used in this study provides a comparable and potentially additive, response to localized VEGF delivery over early time points. These studies demonstrate a materials approach to achieve an angiogenic response formerly limited to the delivery of inductive growth factors.     

Effects of compatibility of deproteinized antler cancellous bone with various bioactive factors on their osteogenic potential

Combinations of calcium phosphate scaffolds and bioactive factors are promising niche-mimetic solutions for repairing large-sized bone defects. However, the importance of compatibility between scaffolds and bioactive factors on their osteogenic outcomes has been largely ignored. This study aimed to investigate the compatibility of calcinated antler cancellous bone (CACB) scaffolds with various bioactive factors including icariin (ICA), velvet antler polypeptides (VAP) or recombinant human bone morphogenetic protein-2 (rhBMP-2) as well as their combinational osteogenic potential in vitro and in vivo. Scanning electron microscopy and fourier transform infrared spectroscopy confirmed the uniform distribution and chemical stability of the reagents on CABC. In vitro release profiles showed relative steady release of ICA from ICA/CACB, burst VAP release from VAP/CACB, and minimal rhBMP-2 release from rhBMP-2/CACB composites. When compared with VAP and rhBMP-2, incorporation of ICA within CACB resulted in most increased cell attachment, proliferation, alkaline phosphatase activity, osteogenic gene expression, and mineralization of rat bone marrow mesenchymal stem cells. In rabbit mandible critical-sized defects, the most extensive osteogenesis and neovascularization were observed in the ICA/CACB group. Differences between the VAP/CACB and rhBMP-2/CACB groups were not apparent. Interestingly, low pro-inflammatory (TNF-α, IL-6) and high anti-inflammatory (IL-10) mRNA levels were observed at scaffold implantation sites which were in close association with amount of new bone formation. These findings highlight that the compatibility between scaffolds and bioactive factors should been taken into account when considering the formula of optimized bone defect repair.     

Reparative Osteogenesis during Transplantation of Mesenchymal Stem Cells

Reparative osteogenesis was studied after xenotransplantation of suspension cell graft from human mesenchymal stem cells. A model of experimental damage to rat femoral diaphysis was developed. The state of animals was satisfactory and non-depressed in the early and late postoperation period. We revealed no local pathological reactions and complications. Administration of mesenchymal stem cells into the area of bone defect accelerated and improved regeneration. Unilateral transplantation of the cell graft stimulated regeneration in the contralateral limb due to acceleration of bone tissue maturation. On day 90 after treatment the bone regenerate was completely developed in the area of defect in animals of various groups. The newly formed bone tissue was well integrated into the bone organ. __________ Translated from Byulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 140, No. 7, pp. 109–113, July, 2005     

Biodegradable,phosphate-containing,dual-gelling macromers for cellular delivery in bone tissue engineering

Injectable, biodegradable, dual-gelling macromer solutions were used to encapsulate mesenchymal stem cells (MSCs) within stable hydrogels when elevated to physiologic temperature. Pendant phosphate groups were incorporated in the N-isopropyl acrylamide-based macromers to improve biointegration and facilitate hydrogel degradation. The MSCs were shown to survive the encapsulation process, and live cells were detected within the hydrogels for up to 28 days in vitro. Cell-laden hydrogels were shown to undergo significant mineralization in osteogenic medium. Cell-laden and acellular hydrogels were implanted into a critical-size rat cranial defect for 4 and 12 weeks. Both cell-laden and acellular hydrogels were shown to degrade in vivo and help to facilitate bone growth into the defect. Improved bone bridging of the defect was seen with the incorporation of cells, as well as with higher phosphate content of the macromer. Furthermore, direct bone-to-hydrogel contact was observed in the majority of implants, which is not commonly seen in this model. The ability of these macromers to deliver stem cells while forming in situ and subsequently degrade while facilitating bone ingrowth into the defect makes this class of macromers a promising material for craniofacial bone tissue engineering.     

Enhanced osteoporotic bone regeneration by strontium-substituted calcium silicate bioactive ceramics

The regeneration capacity of the osteoporotic bones is generally lower than that of the normal bones. Current methods of bone defect treatment for osteoporosis are not always satisfactory. Recent studies have shown that the silicate based biomaterials can stimulate osteogenesis and angiogenesis due to the silicon (Si) ions released from the materials, and enhance bone regeneration in vivo. Other studies showed that strontium (Sr) plays a distinct role on inhibiting bone resorption. Based on the hypothesis that the combination of Si and Sr may have synergetic effects on osteoporotic bone regeneration, the porous Sr-substituted calcium silicate (SrCS) ceramic scaffolds combining the functions of Sr and Si elements were developed with the goals to promote osteoporotic bone defect repair. The effects of the ionic extract from SrCS on osteogenic differentiation of bone marrow mesenchymal stem cells derived from ovariectomized rats (rBMSCs-OVX), angiogenic differentiation of human umbilical vein endothelial cells (HUVECs) were investigated. The in vitro results showed that Sr and Si ions released from SrCS enhanced cell viability, alkaline phosphatase (ALP) activity, and mRNA expression levels of osteoblast-related genes of rBMSCs-OVX and expression of vascular endothelial growth factor (VEGF) without addition of extra osteogenic and angiogenic reagents. The activation in extracellular signal-related kinases (ERK) and p38 signaling pathways were observed in rBMSCs-OVX cultured in the extract of SrCS, and these effects could be blocked by ERK inhibitor PD98059, and P38 inhibitor SB203580, respectively. Furthermore, the ionic extract of SrCS stimulated HUVECs proliferation, differentiation and angiogenesis process. The in vivo experiments revealed that SrCS dramatically stimulated bone regeneration and angiogenesis in a critical sized OVX calvarial defect model, and the enhanced bone regeneration might be attributed to the modulation of osteogenic differentiation of endogenous mesenchymal stem cells (MSCs) and the inhibition of osteoclastogenesis, accompanying with the promotion of the angiogenic activity of endothelial cells (ECs).     

Effect of bioactive borate glass microstructure on bone regeneration,angiogenesis, and hydroxyapatite conversion in a rat calvarial defect model

Borate bioactive glasses are biocompatible and enhance new bone formation, but the effect of their microstructure on bone regeneration has received little attention. In this study scaffolds of borate bioactive glass (1393B3) with three different microstructures (trabecular, fibrous, and oriented) were compared for their capacity to regenerate bone in a rat calvarial defect model. 12 weeks post-implantation the amount of new bone, mineralization, and blood vessel area in the scaffolds were evaluated using histomorphometric analysis and scanning electron microscopy. The amount of new bone formed was 33%, 23%, and 15%, respectively, of the total defect area for the trabecular, oriented, and fibrous microstructures. In comparison, the percent new bone formed in implants composed of silicate 45S5 bioactive glass particles (250–300 μm) was 19%. Doping the borate glass with copper (0.4 wt.% CuO) had little effect on bone regeneration in the trabecular and oriented scaffolds, but significantly enhanced bone regeneration in the fibrous scaffolds (from 15 to 33%). The scaffolds were completely converted to hydroxyapatite within the 12 week implantation. The amount of hydroxyapatite formed, 22%, 35%, and 48%, respectively, for the trabecular, oriented, and fibrous scaffolds, increased with increasing volume fraction of glass in the as-fabricated scaffold. Blood vessels infiltrated into all the scaffolds, but the trabecular scaffolds had a higher average blood vessel area compared with the oriented and fibrous scaffolds. While all three scaffold microstructures were effective in supporting bone regeneration, the trabecular scaffolds supported more bone formation and may be more promising in bone repair.     

Multiscale patterned transplantable stem cell patches for bone tissue regeneration

Stem cell-based therapy has been proposed as an enabling alternative not only for the treatment of diseases but also for the regeneration of tissues beyond complex surgical treatments or tissue transplantation. In this study, we approached a conceptual platform that can integrate stem cells into a multiscale patterned substrate for bone regeneration. Inspired by human bone tissue, we developed hierarchically micro- and nanopatterned transplantable patches as synthetic extracellular matrices by employing capillary force lithography in combination with a surface micro-wrinkling method using a poly(lactic-co-glycolic acid) (PLGA) polymer. The multiscale patterned PLGA patches were highly flexible and showed higher tissue adhesion to the underlying tissue than did the single nanopatterned patches. In response to the anisotropically multiscale patterned topography, the adhesion and differentiation of human mesenchymal stem cells (hMSCs) were sensitively controlled. Furthermore, the stem cell patch composed of hMSCs and transplantable PLGA substrate promoted bone regeneration in vivo when both the micro- and nanotopography of the substrate surfaces were synergistically combined. Thus, our study concludes that multiscale patterned transplantable stem cell patches may have a great potential for bone regeneration as well as for various regenerative medicine approaches.     

血管内皮生长因子与纳米晶胶原基骨支架复合修复大鼠股骨缺损

背景：研究证实纳米晶胶原基骨复合间充质干细胞修复骨缺损具有体内成骨能力。 目的：观察血管内皮生长因子与骨髓间充质干细胞、纳米晶胶原基骨复合物修复大鼠股骨缺损的效果。 方法：制作SD大鼠股骨中段骨缺损模型,随机分为2组：对照组植入骨髓间充质干细胞/纳米晶胶原基骨复合物;实验组植入血管内皮生长因子/骨髓间充质干细胞/纳米晶胶原基骨复合物。术后第2,4,8周行股骨标本影像学与组织学观察;术后第8周行新生骨痂环境扫描电镜检查。 结果与结论：纳米晶胶原基骨支架复合物植入大鼠体内后无排斥反应及炎症反应,且血管内皮生长因子/骨髓间充质干细胞/纳米晶胶原基骨复合物成骨更快,较骨髓间充质干细胞/纳米晶胶原基骨复合物具有更好的骨再生能力,其成骨方式主要为软骨内成骨。推测血管内皮生长因子促进了局部微血管的形成和成骨细胞的分化、增殖,加快了软骨内成骨的速率,缩短了骨修复时间,提高了骨再生的质量和速率。     

可降解聚羟基丁酸酯-羟基戊酸酯的共聚物膜

背景：聚羟基丁酸/羟基戊酸共聚酯已用于构建心脏生物瓣膜,但不知是否可作为骨再生引导膜？ 目的：观察聚羟基丁酸/羟基戊酸共聚酯膜的生物相容性,并评价其成骨能力。 方法：采用MTT法检测100%,75%,50%,25%聚羟基丁酸/羟基戊酸共聚酯材料浸提液对犬骨髓间充质干细胞相对增殖度的影响,并评价其细胞毒性;在犬胫骨缺损模型左右两侧远心端骨缺损上方覆盖聚羟基丁酸/羟基戊酸共聚酯膜作为实验组,在近心端仅以骨膜瓣复位作为对照组。 结果与结论：100%,75%,50%,25%聚羟基丁酸/羟基戊酸共聚酯膜的细胞毒性分级为0~1级,对骨髓间充质干细胞的生长及增殖无毒性;实验组术后第2周即可见新骨形成,12周时骨缺损区已完全被新骨充填,骨修复质量明显优于对照组。表明聚羟基丁酸/羟基戊酸共聚酯膜具有良好的生物相容性,对骨髓间充质干细胞无明显毒性,引导成骨能力强。     

Simultaneous regeneration of articular cartilage and subchondral bone induced by spatially presented TGF-beta and BMP-4 in a bilayer affinity binding system

Subchondral defect repair is a multitask challenge requiring the simultaneous regeneration of cartilage and bone. Herein, we describe the features of a hydrogel system designed to simultaneously induce the endogenous regeneration of hyaline cartilage and subchondral bone. The system was constructed as two layers, spatially presenting the chondroinductive transforming growth factor-β1 (TGF-β1) in one layer and the osteoinductive bone morphogenetic protein-4 (BMP-4) in a second layer, via affinity binding to the matrix. Human mesenchymal stem cells seeded in the bilayer system differentiated into chondrocytes and osteoblasts in the respective layers, confirming the spatial presentation and prolonged activity of TGF-β1 and BMP-4. Administration of the bilayer system with affinity-bound TGF-β1 and BMP-4 (with no cells) into a subchondral defect in rabbits induced endogenous regeneration of articular cartilage and the subchondral bone underneath within 4weeks. Cartilage extracellular matrix proteoglycans were found in the top layer, with no mineralization, whereas the layer underneath consisted of newly formed woven bone. The results indicate that stem cells migrating into the defect are able to sense the biological cues spatially presented in the hydrogel and respond by differentiation into the appropriate cell lineage. The strategy has a real translational potential for repairing osteochondral defects in humans as it is acellular and can be implanted via a minimally invasive method.     

Paper-based bioactive scaffolds for stem cell-mediated bone tissue engineering

Bioactive, functional scaffolds are required to improve the regenerative potential of stem cells for tissue reconstruction and functional recovery of damaged tissues. Here, we report a paper-based bioactive scaffold platform for stem cell culture and transplantation for bone reconstruction. The paper scaffolds are surface-engineered by an initiated chemical vapor deposition process for serial coating of a water-repellent and cell-adhesive polymer film, which ensures the long-term stability in cell culture medium and induces efficient cell attachment. The prepared paper scaffolds are compatible with general stem cell culture and manipulation techniques. An optimal paper type is found to provide structural, physical, and mechanical cues to enhance the osteogenic differentiation of human adipose-derived stem cells (hADSCs). A bioactive paper scaffold significantly enhances in vivo bone regeneration of hADSCs in a critical-sized calvarial bone defect. Stacking the paper scaffolds with osteogenically differentiated hADSCs and human endothelial cells resulted in vascularized bone formation in vivo. Our study suggests that paper possesses great potential as a bioactive, functional, and cost-effective scaffold platform for stem cell-mediated bone tissue engineering. To the best of our knowledge, this is the first study reporting the feasibility of a paper material for stem cell application to repair tissue defects.     

Comparative evaluation of MSCs from bone marrow and adipose tissue seeded in PRP-derived scaffold for cartilage regeneration

The aims of this study were to (1) determine whether platelet-rich plasma (PRP) could be prepared as a bioactive scaffold capable of endogenous growth factor release for cartilage repair; (2) compare the chondrogenic differentiation ability of mesenchymal stem cells (MSCs) from bone marrow (BMSC) and from adipose (ADSC) seeded within the PRP scaffold; and (3) test the efficacy of ADSC-PRP construct in cartilage regeneration in?vivo. In?vitro evaluation showed that a 3-dimensional scaffold with a mesh-like microstructure was formed from PRP, with the capability of endogenous growth factor release and ready cell incorporation. Upon seeding in the PRP scaffold, BMSC showed higher proliferation rate, and higher expression of cartilage-specific genes and proteins than ADSC. In an osteochondral defect model in rabbits, implanted BMSC seeded within PRP scaffold also exhibited better gross appearance and histological and immunohistochemical characteristics, higher cartilage-specific gene and protein expression as well as subchondral bone regeneration. ADSC seeded constructs developed into functional chondrocytes secreting cartilaginous matrix in rabbits at 9 weeks post-implantation. Our findings suggest that PRP is a candidate bioactive scaffold capable of releasing endogenous growth factors and that BMSC and ADSC seeded within the PRP scaffold differentiate into chondrocytes and may be suitable for cell-based cartilage repair.     

Reconstruction of goat femur segmental defects using triphasic ceramic-coated hydroxyapatite in combination with autologous cells and platelet-rich plasma

Segmental bone defects resulting from trauma or pathology represent a common and significant clinical problem. In this study, a triphasic ceramic (calcium silicate, hydroxyapatite and tricalcium phosphate)-coated hydroxyapatite (HASi) having the benefits of both HA (osteointegration, osteoconduction) and silica (degradation) was used as a bone substitute for the repair of segmental defect (2 cm) created in a goat femur model. Three experimental goat femur implant groups – (a) bare HASi, (b) osteogenic-induced goat bone marrow-derived mesenchymal stem cells cultured HASi (HASi + C) and (c) osteogenic-induced goat bone marrow-derived mesenchymal stem cells cultured HASi + platelet-rich plasma (HASi + CP) – were designed and efficacy performance in the healing of the defect was evaluated. In all the groups, the material united with host bone without any inflammation and an osseous callus formed around the implant. This reflects the osteoconductivity of HASi where the cells have migrated from the cut ends of host bone. The most observable difference between the groups appeared in the mid region of the defect. In bare HASi groups, numerous osteoblast-like cells could be seen together with a portion of material. However, in HASi + C and HASi + CP, about 60–70% of that area was occupied by woven bone, in line with material degradation. The interconnected porous nature (50–500 μm), together with the chemical composition of the HASi, facilitated the degradation of HASi, thereby opening up void spaces for cellular ingrowth and bone regeneration. The combination of HASi with cells and PRP was an added advantage that could promote the expression of many osteoinductive proteins, leading to faster bone regeneration and material degradation. Based on these results, we conclude that bare HASi can aid in bone regeneration but, with the combination of cells and PRP, the sequence of healing events are much faster in large segmental bone defects in weight-bearing areas in goats.     

钠离子通道阻断剂抑制骨髓间充质干细胞向心肌样细胞的分化

背景：研究发现人和动物的多种间充质干细胞均存在河豚毒素敏感性钠离子通道,但钠离子通道是否会影响间充质干细胞向心肌细胞分化尚无共识。 目的：观察钠离子通道特异性阻断剂河豚毒素对骨髓间充质干细胞向心肌样细胞分化的影响。 方法：利用Percoll’s密度梯度离心法结合差速贴壁法分离大鼠骨髓间充质干细胞,用主动脉逆行生物酶灌流法分离大鼠成体心肌细胞。将DAPI标记的大鼠骨髓间充质干细胞与成体心肌细胞混合培养,应用0,10,100 μmol/L的河豚毒素进行干预,1周后观察骨髓间充质干细胞向心肌样细胞的分化情况。 结果与结论：免疫荧光细胞化学染色显示,大鼠骨髓间充质干细胞和成体心肌细胞混合培养1周后,骨髓间充质干细胞显著表达心肌特异性蛋白结蛋白和肌球蛋白,而10,100 μmol/L的河豚毒素均可完全抑制结蛋白和肌球蛋白在骨髓间充质干细胞中的表达。提示钠离子通道是骨髓间充质干细胞向心肌样细胞分化的必要条件。