Silk coating on a bioactive ceramic scaffold for bone regeneration: effective enhancement of mechanical and in vitro osteogenic properties towards load‐bearing applications

Bioactive ceramic scaffolds represent competitive choices for clinical bone reconstruction, but their widespread use is restricted by inherent brittleness and weak mechanical performance under load. This study reports the development of strong and tough bioactive scaffolds suitable for use in load‐bearing bone reconstruction. A strong and bioactive ceramic scaffold (strontium–hardystonite–gahnite) is combined with single and multiple coating layers of silk fibroin to enhance its toughness, producing composite scaffolds which match the mechanical properties of cancellous bone and show enhanced capacity to promote in vitro osteogenesis. Also reported for the first time is a comparison of the coating effects obtained when a polymeric material is coated on ceramic scaffolds with differing microstructures, namely the strontium–hardystonite–gahnite scaffold with high‐density struts as opposed to a conventional ceramic scaffold, such as biphasic calcium phosphate, with low‐density struts. The results show that silk coating on a unique ceramic scaffold can lead to simple and effective enhancement of its mechanical and biological properties to suit a wider range of applications in clinical bone reconstruction, and also establish the influence of ceramic microstructure on the effectiveness of silk coating as a method of reinforcement when applied to different types of ceramic bone graft substitutes. Copyright © 2015 John Wiley & Sons, Ltd.     

Hierarchical starch‐based fibrous scaffold for bone tissue engineering applications

Fibrous structures mimicking the morphology of the natural extracellular matrix are considered promising scaffolds for tissue engineering. This work aims to develop a novel hierarchical starch‐based scaffold. Such scaffolds were obtained by a combination of starch–polycaprolactone micro‐ and polycaprolactone nano‐motifs, respectively produced by rapid prototyping (RP) and electrospinning techniques. Scanning electron microscopy (SEM) and micro‐computed tomography analysis showed the successful fabrication of a multilayer scaffold composed of parallel aligned microfibres in a grid‐like arrangement, intercalated by a mesh‐like structure with randomly distributed nanofibres (NFM). Human osteoblast‐like cells were dynamically seeded on the scaffolds, using spinner flasks, and cultured for 7 days under static conditions. SEM analysis showed predominant cell attachment and spreading on the nanofibre meshes, which enhanced cell retention at the bulk of the composed/hierarchical scaffolds. A significant increment in cell proliferation and osteoblastic activity, assessed by alkaline phosphatase quantification, was observed on the hierarchical fibrous scaffolds. These results support our hypothesis that the integration of nanoscale fibres into 3D rapid prototype scaffolds substantially improves their biological performance in bone tissue‐engineering strategies. Copyright © 2008 John Wiley & Sons, Ltd.     

Human nail bed‐derived decellularized scaffold regulates mesenchymal stem cells for nail plate regeneration

Among hand trauma, nail bed is the most involved tissue in hospital emergency departments, resulting in the loss of nail plate, which leads to a disturbance of hand grasp function, long‐lasting digit tip pain, hyperpathia, and disesthesia. Treatment of nail bed defects is a significant clinical challenge due to the lack of uniform nail bed thickness and distinct regenerative ability. In this study, it is shown that the extracellular matrix of decellularized nail bed scaffolds can play an important role in inducing bone mesenchymal stem cells to differentiate into nail epithelial cells. Using decellularized nail bed scaffolds combined with bone mesenchymal stem cells, it is revealed that the engineered nail bed can promote nude mouse nail plate regeneration ectopically. The natural extracellular matrix of decellularized nail bed scaffolds can serve as a 3D structural template for bone mesenchymal stem cell differentiation into nail‐associated cells, initiating the nail plate regeneration. These results not only provide a proof‐of‐principle for the generation of transplantable nail grafts based on decellularized nail bed scaffolds derived from clinically wasted amputated fingers but also provide important considerations for clinical treatment for digit tip trauma.     

Accelerating bone defects healing in calvarial defect model using 3D cultured bone marrow‐derived mesenchymal stem cells on demineralized bone particle scaffold

Bone defects are usually difficult to be regenerated due to pathological states or the size of the injury. Researchers are focusing on tissue engineering approaches in order to drive the regenerative events, using stem cells to regenerate bone. The purpose of this study is to evaluate the osteogenic differentiation of bone marrow‐derived mesenchymal stem cells (BMSCs) on biologically derived Gallus gallus domesticus‐derived demineralized bone particle (GDD) sponge. The sponges were prepared by freeze‐drying method using 1, 2, and 3 wt% GDD and cross‐linked with glutaraldehyde. The GDD sponge was characterized using scanning electron microscopy, compressive strength, porosity, and Fourier transform infrared. The potential bioactivity of the sponge was evaluated by osteogenic differentiation of BMSCs using 3(4, dimethylthiazol‐2‐yl)‐2, 5‐diphenyltetrazolium bromide assay and quantifying alkaline phosphatase (ALP) activity. in vivo experiments were evaluated through a micro‐computerized tomography (μ ‐CT) and histological assays. The analysis confirmed that an increase in the concentration of the GDD in the sponge leads to a higher bone formation and deposition in rat calvarial defects. Histological assay results were in line with μ ‐CT. The results reported in this study demonstrated the potential application of GDD sponges as osteoinductor in bone tissue engineering in pathological or nonunion bone defects.     

A tissue‐like construct of human bone marrow MSCs composite scaffold support in vivo ectopic bone formation

Biocompatible and osteoconductive cell–scaffold constructs comprise the first and most important step towards successful in vivo bone repair. This study reports on a new cell–scaffold construct composed of gelatin‐based hydrogel and ceramic (CaCO₃/β‐TCP) particles loaded with human MSCs producing a tissue‐like construct applied as a transplant for in vivo bone formation. Bone marrow‐derived human MSCs were cultured in osteogenic induction medium. 5 × 10⁵ (P₂) cells were loaded on a mixture of hydrogel microspheres and ceramic particles, cultured in a rotating dynamic culture for up to 3 weeks. Both hydrogel microspheres and ceramic particles coalesced together to form a tissue‐like construct, shown by histology to contain elongated spindle‐like cells forming the new tissue between the individual particles. Cell proliferation and cell viability were confirmed by Alamar blue assay and by staining with CFDA, respectively. FACS analysis conducted before loading the cells, and after formation of the construct, revealed that the profile of cell surface markers remained unchanged throughout the dynamic culture. The osteogenic potential of the cells composing the tissue‐like construct was further validated by subcutaneous transplants in athymic nude mice. After 8 weeks a substantial amount of new bone formation was observed in the cell‐construct transplants, whereas no bone formation was observed in transplants containing no cells. This new cell construct provides a system for in vivo bone transplants. It can be tailored for a specific size and shape as needed for various transplant sites and for all aspects of regenerative medicine and biomaterial science. Copyright © 2009 John Wiley & Sons, Ltd.     

Bone regeneration in minipigs via calcium phosphate cement scaffold delivering autologous bone marrow mesenchymal stem cells and platelet‐rich plasma

Macroporous calcium phosphate cement (CPC) with stem cell seeding is promising for bone regeneration. The objective of this study was to investigate the effects of co‐delivering autologous bone marrow mesenchymal stem cells (BMSCs) and autologous platelet‐rich plasma (PRP) in CPC scaffold for bone regeneration in minipigs for the first time. Twelve female adult Tibet minipigs (12–18 months old) were used. A cylindrical defect with 10 mm height and 8 mm diameter was prepared at the femoral condyle. Two bone defects were created in each minipig, one at each side of the femoral condyle. Three constructs were tested: (1) CPC scaffold (CPC control); (2) CPC seeded with BMSCs (CPC‐BMSC); (3) CPC seeded with BMSCs and PRP (CPC‐BMSC‐PRP). Two time points were tested: 6 and 12 weeks (n = 4). Good integration of implant with surrounding tissues was observed in all groups. At 12 weeks, the CPC‐BMSC‐PRP group had significantly less residual CPC remaining in the defect than the CPC‐BMSC group and the CPC control (p < 0.05). The residual CPC volume for the CPC‐BMSC‐PRP group was half that of the CPC control. New bone formation for CPC‐BMSC‐PRP was more than two‐fold that of the CPC control (p < 0.05). CPC‐BMSC‐PRP had new blood vessel density that was nearly two‐fold that of the CPC control (p < 0.05). In conclusion, CPC scaffold with autologous BMSC‐PRP doubled the new bone regeneration and blood vessel density in minipigs compared with the CPC control. In the present study, the new macroporous CPC system with co‐delivered BMSC‐PRP has been shown to promote scaffold resorption and bone regeneration in large defects. Copyright © 2017 John Wiley & Sons, Ltd.     

Engineering axially vascularized bone in the sheep arteriovenous‐loop model

Treatment of complex bone defects in which vascular supply is insufficient is still a challenge. To overcome the limitations from autologous grafts, a sheep model has been established recently, which is characterized by the development of an independent axial vascularization of a bioartificial construct, permitting microsurgical transplantation. To engineer independently axially vascularized bone tissue in the sheep arteriovenous (AV)‐loop model, mesenchymal stem cells (MSCs), without and in combination with recombinant human bone morphogenetic protein‐2 (rhBMP‐2), were harvested and directly autotransplanted in combination with β‐tricalcium phosphate–hydroxyapatite (β‐TCP–HA) granules into sheep in this study. After explantation after 12 weeks, histological and immunohistochemical evaluation revealed newly formed bone in both groups. An increased amount of bone area was obtained using directly autotransplanted MSCs with rhBMP‐2 stimulation. Osteoblastic and osteoclastic cells were detected adjacent to the newly formed bone, revealing an active bone remodelling process. Directly autotransplanted MSCs can be found close to the β‐TCP–HA granules and are contributing to bone formation. Over time, magnetic resonance imaging (MRI) and micro‐computed tomography (μCT) imaging confirmed the dense vascularization arising from the AV‐loop. This study shows de novo engineering of independently axially vascularized transplantable bone tissue in clinically significant amounts, using directly autotransplanted MSCs and rhBMP‐2 stimulation in about 12 weeks in the sheep AV‐loop model. This strategy of engineering vascularized transplantable bone tissue could be possibly transferred to the clinic in the future in order to augment current reconstructive strategies. Copyright © 2012 John Wiley & Sons, Ltd.     

Perfusion conditioning of hydroxyapatite-chitosan-gelatin scaffolds for bone tissue regeneration from human mesenchymal stem cells

Tissue-engineered bone grafts require an osteogenic cell source and a scaffold capable of supporting tissue regeneration. Hydroxyapatite (H), chitosan (C), and gelatin (G), when combined, produce a biomimetic scaffold with a chemical similarity to the main structural components of natural bone tissue. In this study a phase-separation technique was used to produce a porous 3D HCG scaffold, containing a network of cross-linked chitosan and gelatin fibrils coated in hydroxyapatite, with pore size readily controlled by freezing temperature. The HCG scaffolds were then seeded with human mesenchymal stem cells (hMSCs), using a depth filtration system after preconditioning with serum-containing medium for 7 days under either static or perfusion conditions. The effects of static and perfusion media preconditioning on protein adsorption, surface morphology, hMSC attachment, proliferation and osteogenic differentiation were examined. Perfusion preconditioning, as opposed to static preconditioning, enhances adsorption of ECM proteins, which in turn promotes hMSC proliferation and osteogenic differentiation. The results demonstrate the importance of convective flow in modulating the 3D HCG microenvironment and highlight its profound influence on 3D construct development.     

Sequential identification of a degradable phosphate glass scaffold for skeletal muscle regeneration

Tissue engineering has the potential to overcome limitations associated with current management of skeletal muscle defects. This study aimed to sequentially identify a degradable phosphate glass scaffold for the restoration of muscle defects. A series of glass compositions were investigated for the potential to promote bacterial growth. Thereafter, the response of human craniofacial muscle‐derived cells was determined. Glass compositions containing Fe4‐ and 5 mol% did not promote greater Staphylococcus aureus and Staphylococcus epidermidis growth compared to the control (p > 0.05). Following confirmation of myogenicity, further studies assessed the biocompatibility of glasses containing Fe5 mol%. Cells seeded on collagen‐coated disks demonstrated comparable cellular metabolic activity to control. Upregulation of genes encoding for myogenic regulatory factors (MRFs) confirmed myofibre formation and there was expression of developmental MYH genes. The use of 3‐D aligned fibre scaffolds supported unidirectional cell alignment and upregulation of MRF and developmental MYH genes. Compared to the 2‐D disks, there was also expression of MYH2 and MYH7 genes, indicating further myofibre maturation on the 3‐D scaffolds and confirming the importance of key biophysical cues. Copyright © 2012 John Wiley & Sons, Ltd.     

Substance P/dexamethasone‐encapsulated PLGA scaffold fabricated using supercritical fluid process for calvarial bone regeneration

Poly(lactic‐co‐glycolic acid) (PLGA) scaffolds encapsulated with substance P (SP) and dexamethasone (Dex) by the supercritical CO₂ foaming method were fabricated to treat calvarial bone. We compared the release profiles of SP and Dex according to the incorporation methods using encapsulation or dipping. Ninety percent of the SP or Dex molecules in the scaffolds prepared by the encapsulating method were released by day 14 or day 6, respectively. In vivo real‐time assays for human mesenchymal stem cell (hMSC) tracking were performed to confirm the MSC recruitment abilities of the scaffolds. The results showed that the optical intensity of the SP‐encapsulated group was 2.59 times higher than that of the phosphate‐buffered saline group and 1.3 times higher than that of the SP‐dipping group. Furthermore, we compared the angiogenesis activity of the scaffolds. In the SP‐encapsulated group, 72.9  ±  2.6% of the vessels showed matured features by 1 week, and it increased to 82.0  ±  4.6% after 4 weeks. We implanted the scaffolds into rat calvarial defects. After 24 weeks, SP‐ and Dex‐encapsulated scaffolds showed 67.1% and 26.2% higher bone formation than those of the Dex‐encapsulated group and SP‐encapsulated group, respectively, and they formed 36.1% more bone volume compared with the SP‐ and Dex‐dipped scaffolds. Consequently, the results of this study suggest that SP‐ and Dex‐encapsulated scaffolds made by the supercritical CO₂ foaming method could be a good treatment modality to treat critical bone defects without cell transplantation by recruiting autologous stem cells and forming new bone tissues. Copyright © 2017 John Wiley & Sons, Ltd.     

Human adipose‐derived cells can serve as a single‐cell source for the in vitro cultivation of vascularized bone grafts

Orthopaedic surgery often requires bone grafts to correct large defects resulting from congenital defects, surgery or trauma. Great improvements have been made in the tissue engineering of bone grafts. However, these grafts lack the vascularized component that is critical for their survival and function. From a clinical perspective, it would be ideal to engineer vascularized bone grafts starting from one single‐cell harvest obtained from the patient. To this end, we explored the potential of human adipose‐derived mesenchymal stem cells (hASCs) as a single‐cell source for osteogenic and endothelial differentiation and the assembly of bone and vascular compartments within the same scaffold. hASCs were encapsulated in fibrin hydrogel as an angioinductive material for vascular formation, combined with a porous silk fibroin sponge to support osteogenesis, and subjected to sequential application of growth factors. Three strategies were evaluated by changing spatiotemporal cues: (a) induction of osteogenesis prior to vasculogenesis; (b) induction of vasculogenesis prior to osteogenesis; or (c) simultaneous induction of osteogenesis and vasculogenesis. By 5 weeks of culture, bone‐like tissue development was evidenced by the deposition of bone matrix proteins, alkaline phosphatase activity and calcium deposition, along with the formation of vascular networks, evidenced by endothelial cell surface markers, such as CD31 and von Willebrand factor, and morphometric analysis. Most robust development of the two tissue compartments was achieved by sequential induction of osteogenesis followed by the induction of vasculogenesis. Taken together, the collected data strongly support the utility of hASCs as a single‐cell source for the formation of vascularized bone tissue. Copyright © 2012 John Wiley & Sons, Ltd.     

A new composite hydrogel combining the biological properties of collagen with the mechanical properties of a supramolecular scaffold for bone tissue engineering

Tissue engineering is a promising alternative to autografts, allografts, or biomaterials to address the treatment of severe and large bone lesions. Classically, tissue engineering products associate a scaffold and cells and are implanted or injected into the lesion. These cells must be embedded in an appropriate biocompatible scaffold, which offers a favourable environment for their survival and differentiation. Here, we designed a composite hydrogel composed of collagen I, an extracellular matrix protein widely used in several therapeutic applications, which we associated with a physical hydrogel generated from a synthetic small amphiphilic molecule. This composite showed improved mechanical and biological characteristics as compared with gels obtained from each separate compound. Incorporation of the physical hydrogel prevented shrinkage of collagen and cell diffusion out of the gel and yielded a gel with a higher elastic modulus than those of gels obtained with each component alone. The composite hydrogel allowed cell adhesion and proliferation in vitro and long‐term cell survival in vivo. Moreover, it promoted the differentiation of human adipose‐derived stem cells in the absence of any osteogenic factors. In vivo, cells embedded in the composite gel and injected subcutaneously in immunodeficient mice produced lamellar osteoid tissue and differentiated into osteoblasts. This study points this new composite hydrogel as a promising scaffold for bone tissue engineering applications.     

A collagen‐coated sponge silk scaffold for functional meniscus regeneration

Tissue engineering is a promising solution for meniscal regeneration after meniscectomy. However, in situ reconstruction still poses a formidable challenge due to multifunctional roles of the meniscus in the knee. In this study, we fabricate a silk sponge from 9% (w/v) silk fibroin solution through freeze drying and then coat its internal space and external surface with collagen sponge. Subsequently, various characteristics of the silk‐collagen scaffold are evaluated, and cytocompatibility of the construct is assessed in vitro and subcutaneously. The efficacy of this composite scaffold for meniscal regeneration is evaluated through meniscus reconstruction in a rabbit meniscectomy model. It is found that the internally coated collagen sponge enhances the cytocompatibility of the silk sponge, and the external layer of collagen sponge significantly improves the initial frictional property. Additionally, the silk‐collagen composite group shows more tissue ingrowth and less cartilage wear than the pure silk sponge group at 3 months postimplantation in situ. These findings thus demonstrate that the composite scaffold had less damage to the joint surface than the silk alone through promoting functional meniscal regeneration after meniscectomy, which indicates its clinical potential in meniscus reconstruction.     

Large animal in vivo evaluation of a binary blend polymer scaffold for skeletal tissue‐engineering strategies; translational issues

Binary blend polymers offer the opportunity to combine different desirable properties into a single scaffold, to enhance function within the field of tissue engineering. Previous in vitro and murine in vivo analysis identified a polymer blend of poly(l ‐lactic acid)–poly(ε‐caprolactone) (PLLA:PCL 20:80) to have characteristics desirable for bone regeneration. Polymer scaffolds in combination with marrow‐derived skeletal stem cells (SSCs) were implanted into mid‐shaft ovine 3.5 cm tibial defects, and indices of bone regeneration were compared to groups implanted with scaffolds alone and with empty defects after 12 weeks, including micro‐CT, mechanical testing and histological analysis. The critical nature of the defect was confirmed via all modalities. Both the scaffold and scaffold/SSC groups showed enhanced quantitative bone regeneration; however, this was only found to be significant in the scaffold/SSCs group (p = 0.04) and complete defect bridging was not achieved in any group. The mechanical strength was significantly less than that of contralateral control tibiae (p < 0.01) and would not be appropriate for full functional loading in a clinical setting. This study explored the hypothesis that cell therapy would enhance bone formation in a critical‐sized defect compared to scaffold alone, using an external fixation construct, to bridge the scale‐up gap between small animal studies and potential clinical translation. The model has proved a successful critical defect and analytical techniques have been found to be both valid and reproducible. Further work is required with both scaffold production techniques and cellular protocols in order to successfully scale‐up this stem cell/binary blend polymer scaffold. © 2015 The Authors. Journal of Tissue Engineering and Regenerative Medicine published by John Wiley & Sons, Ltd.     

骨髓间充质干细胞与脱细胞脑组织支架的生物相容性

背景：骨髓间充质干细胞与细胞载体联合移植治疗中枢神经系统损伤还处于实验研究阶段。目的：评价大鼠骨髓间充质干细胞与脱细胞脑组织支架的生物相容性,探讨脱细胞脑组织支架作为中枢神经系统组织工程材料的可行性。方法：以全骨髓法分离纯化大鼠骨髓间充质干细胞,通过物理及化学方法相结合制各脱细胞脑组织支架。将转染携带绿色荧光蛋白基因的骨髓间充质干细胞种植到支架材料上共培养,通过倒置相差显微镜、扫描电镜、激光共聚焦显微镜等方法观察脱细胞脑组织支架的内部结构及复合支架上细胞的生长状况。结果与结论：制备的脱细胞脑组织支架材料呈三维立体网状结构。骨髓间充质干细胞可在支架上黏附生长,形态良好。大鼠骨髓间充质干细胞与脱细胞脑组织支架具有良好的生物相容性,有望作为中枢神经系统组织工程的载体材料。     

Bone regeneration by means of a three‐dimensional printed scaffold in a rat cranial defect

Recently, computer‐designed three‐dimensional (3D) printing techniques have emerged as an active research area with almost unlimited possibilities. In this study, we used a computer‐designed 3D scaffold to drive new bone formation in a bone defect. Poly‐L‐lactide (PLLA) and bioactive β‐tricalcium phosphate (TCP) were simply mixed to prepare ink. PLLA + TCP showed good printability from the micronozzle and solidification within few seconds, indicating that it was indeed printable ink for layer‐by‐layer printing. In the images, TCP on the surface of (and/or inside) PLLA in the printed PLLA + TCP scaffold looked dispersed. MG‐63 cells (human osteoblastoma) adhered to and proliferated well on the printed PLLA + TCP scaffold. To assess new bone formation in vivo, the printed PLLA + TCP scaffold was implanted into a full‐thickness cranial bone defect in rats. The new bone formation was monitored by microcomputed tomography and histological analysis of the in vivo PLLA + TCP scaffold with or without MG‐63 cells. The bone defect was gradually spontaneously replaced with new bone tissues when we used both bioactive TCP and MG‐63 cells in the PLLA scaffold. Bone formation driven by the PLLA + TCP30 scaffold with MG‐63 cells was significantly greater than that in other experimental groups. Furthermore, the PLLA + TCP scaffold gradually degraded and matched well the extent of the gradual new bone formation on microcomputed tomography. In conclusion, the printed PLLA + TCP scaffold effectively supports new bone formation in a cranial bone defect.     

Short‐term evaluation of electromagnetic field pretreatment of adipose‐derived stem cells to improve bone healing

An electromagnetic field is an effective stimulation tool because it promotes bone defect healing, albeit in an unknown way. Although electromagnetic fields are used for treatment after surgery, many patients prefer cell‐based tissue regeneration procedures that do not require daily treatments. This study addressed the effects of an electromagnetic field on adipose‐derived stem cells (ASCs) to investigate the feasibility of pretreatment to accelerate bone regeneration. After identifying a uniform electromagnetic field inside a solenoid coil, we observed that a 45 Hz electromagnetic field induced osteogenic marker expression via bone morphogenetic protein, transforming growth factor β, and Wnt signalling pathways based on microarray analyses. This electromagnetic field increased osteogenic gene expression, alkaline phosphate activity and nodule formation in vitro within 2 weeks, indicating that this pretreatment may provide osteogenic potential to ASCs on three‐dimensional (3D) ceramic scaffolds. This pretreatment effect of an electromagnetic field resulted in significantly better bone regeneration in a mouse calvarial defect model over 4 weeks compared to that in the untreated group. This short‐term evaluation showed that the electromagnetic field pretreatment may be a future therapeutic option for bone defect treatment. Copyright © 2012 John Wiley & Sons, Ltd.     

A novel three‐dimensional scaffold for regenerative endodontics: materials and biological characterizations

An electrospun nanocomposite fibrous material holds promise as a scaffold, as well as a drug‐delivery device to aid in root maturogenesis and the regeneration of the pulp–dentine complex. A novel three‐dimensional (3D) nanocomposite scaffold composed of polydioxanone (PDS II®) and halloysite nanotubes (HNTs) was designed and fabricated by electrospinning. Morphology, structure, mechanical properties and cell compatibility studies were carried out to evaluate the effects of HNTs incorporation (0.5–10 wt% relative to PDS w/w). Overall, a 3D porous network was seen in the different fabricated electrospun scaffolds, regardless of the HNT content. The incorporation of HNTs at 10 wt% led to a significant (p < 0.0001) fibre diameter increase and a reduction in scaffold strength. Moreover, PDS–HNTs scaffolds supported the attachment and proliferation of human‐derived pulp fibroblast cells. Quantitative proliferation assay performed with human dental pulp‐derived cells as a function of nanotubes concentration indicated that the HNTs exhibit a high level of biocompatibility, rendering them good candidates for the potential encapsulation of distinct bioactive molecules. Collectively, the reported data support the conclusion that PDS–HNTs nanocomposite fibrous structures hold potential in the development of a bioactive scaffold for regenerative endodontics. Copyright © 2013 John Wiley & Sons, Ltd.     

同种异体脂肪干细胞复合脱钙骨支架材料修复兔胫骨缺损的实验研究

目的探讨同种异体脂肪干细胞复合脱钙骨支架材料修复兔胫骨缺损的实验研究。方法购自北京生物有限公司(动物实验中心)新西兰大白兔8对,其中2对白兔进行脂肪干细胞分离培养,并采用反复滴加方法将细胞均匀分布于支架材料上,获取脂肪干细胞复合脱钙骨材料。其中6对构建兔胫骨骨缺损模型,造模后即刻将单纯脱钙骨材料植入右侧缺损区设为对照组,将诱导后的兔脂肪干细胞-脱钙骨支架复合物植入左侧缺损区设为实验组。于术后12周采用空气栓塞处死大白兔,获取大白兔双侧后肢胫骨修复骨及其周围软组织,行Micro-CT和组织学检测,了解实验组和对照组白兔骨组织生长情况,兔脂肪干细胞-脱钙骨材料成骨表型测定体内降解情况和组织学检测结果。结果大体观察显示实验组白兔后肢骨缺损区可见骨样组织修复,对照组白兔为纤维样组织充填未见骨样组织覆盖;三维CT影像学显示实验组白兔骨缺损断端和材料接合部已充分融合且骨轮廓、光滑的骨塑形清晰可见,对照组白兔骨缺损处多为纤维性连接;HE染色下可见实验组白兔有典型再生骨组织、大量骨陷窝、骨细胞及部分骨小梁结构,对照组白兔为胶原纤维样组织和残留脱钙骨基质、无大片再生骨样组织。两组白兔Ⅱ型胶原免疫组织化学染色均呈阳性,细胞胞浆及胞外基质呈棕黄色,胞核呈蓝色,证实修复细胞表达Ⅱ型胶原,为软骨细胞。实验组和对照组白兔修复组织评分分别为(2.41±0.40)、(2.70±0.45)分,实验组白兔组织修复得分高于对照组白兔,差异有显著性(P<0.05)。结论兔脂肪干细胞接种于脱钙骨材料,经成骨诱导后具有较强成骨分化能力,能较好地修复兔胫骨骨缺损,具有较高临床疗效。