Functional synergy of anti-mir221 and nanohydroxyapatite scaffold in bone tissue engineering of rat skull

An appropriate cell source, effective cell modification, and proper supportive matrices are the main bases of tissue engineering. The effectiveness of anti-mir221 or hydroxyapatite (HA) in improving the osteogenic differentiation of mesenchymal stem cells (MSCs) has been reported previously. Herein, simultaneous application of these osteogenic inducers was investigated in vivo. The Poly-caprolactone (PCL)/HA nanofibers were characterized using contact angle measurement, tensile test, Fourier transform infrared spectroscopy, and electron microscopy. Rat MSCs were isolated, characterized and transfected with anti-mir221. The rats were divided into 4 groups and an 8 mm defect were created in the mid-calvaria of each rat by trephine bur. Group 1 received (PCL)/HA nanofibers, group 2 received (PCL)/HA nanofibers plus autologous MSCs, group 3 received (PCL)/HA nanofibers plus MSCs transfected with anti-mir221, and group 4 rats were left empty as an additional control group. Histomorphometric and radiomorphometric evaluation after 4 and 8 weeks revealed more new bone formation in the cell-treated groups compared to the scaffold alone group. There was evidence for a combination of increased osteoclasts and osteoblast vascular lake containing red blood cells in the anti-mir221 transfected group. New bone penetration into the scaffolds empirically demonstrated the capability of this combination for efficient osteointegration. Altogether, the co-application of HA and anti-mir221 transfected cells can enhance bone healing of the rat skull.     

A novel PHBV/HA microsphere releasing system loaded with alendronate

Microspheres of poly(β-hydroxybutyrate-co-β-hydroxyvalerate) (PHBV) incorporated with hydroxyapatite (HA) and loaded with alendronate (AL), an osteoporosis preventing drugs, were prepared by single emulsion technique. Several methods were used to evaluate this novel drug carrier microsphere system (referred as PHBV/HA–AL). Fourier transform infrared (FTIR) was used to evaluate the enwrapping of HA and the X-ray diffraction (XRD) analysis further confirmed the success. The morphology of PHBV/HA–AL microspheres was observed by scanning electron microscope (SEM), showing rough surface with HA particles enwrapped in the PHBV matrix. The in vitro drug releasing profile of PHBV/HA–AL system was investigated in a 26-day period. There is a sustained releasing pattern after a slight burst release during the first few days. Additionally, rabbit mesenchymal stem cells (MSCs) were used to evaluate the cytotoxicity of the PHBV/HA–AL composites. This controlled release system can well support the proliferation of MSCs. The novel PHBV/HA–AL controlled release system is promising for bone repair therapy.     

中空羟基磷灰石微球作为rhBMP-2缓释载体的研究

利用锂钙硼玻璃在磷酸盐溶液中的原位转化反应制备表面多孔且中空的羟基磷灰石(HA)微球, 将重组人骨形态发生蛋白2(rhBMP-2)装载到微球中, 研究了微球中rhBMP-2的体外缓释行为, 并采用体外细胞培养技术, 将载有rhBMP-2的微球和大鼠骨髓间充质干细胞(MSCs)一起培养, 测定细胞的碱性磷酸酶(ALP)活性, 并与单纯rhBMP-2的作用进行比较. 结果显示, 微球中所装载的rhBMP-2具有明显的缓释效应, 体外释放周期达到1000h以上, 该微球缓释系统具有一定的生物活性, 其作用效果优于单纯使用rhBMP-2.     

In vivo bone regeneration with injectable chitosan/hydroxyapatite/collagen composites and mesenchymal stem cells

For reconstruction of irregular bone defects, injectable biomaterials are more appropriate than the preformed biomaterials. We herein develop a biomimetic in situ-forming composite consisting of chitosan (CS) and mineralized collagen fibrils (nHAC), which has a complex hierarchical structure similar to natural bone. The CS/nHAC composites with or without mesenchymal stem cells (MSCs) are injected into cancellous bone defects at the distal end of rabbit femurs. Defects are assessed by radiographic, histological diagnosis and Raman microscopy until 12 weeks. The results show that MSCs improve the biocompatibility of CS/nHAC composites and enhance new bone formation in vivo at 12 weeks. It can be concluded that the injectable CS/nHAC composites combined with MSCs may be a novel method for reconstruction of irregular bone defects.     

羟基磷灰石球形颗粒表面微形貌构建及其对干细胞生物学行为的调控

通过调节溶胶-凝胶体系中羟基磷灰石(HA)粉末和甲壳素(Chitin)的质量比, 制备具有不同表面微形貌的HA球形颗粒。扫描电子显微镜(SEM)表征结果显示: 随着HA/Chitin质量比从4/1增加到35/1, 球形颗粒的表面微形貌发生了明显变化, 由粗糙渐趋平滑, 微米级皱褶逐渐减少至消失, 微孔隙率从(35%±0.8%)减少到(10.4%±0.7%)。体外细胞培养的结果表明具有微米级皱褶, 微孔隙率较高的粗糙表面具有引导干细胞铺展和增殖的作用, 微孔隙率低的平滑表面则具有引导干细胞轴向延伸及骨向分化的趋势。同时, HA球形颗粒表面微形貌对干细胞表面特征性抗原标志物的表达具有调控作用。     

Triphasic ceramic coated hydroxyapatite as a niche for goat stem cell-derived osteoblasts for bone regeneration and repair

Current treatment strategies for the repair or replacement of bone use synthetic implants with stem cells and their progeny––a new approach to address unmet medical needs. This study has evaluated the effect of a silica-coated bioactive ceramic, namely HASi in comparison to hydroxyapatite (HA) on the adhesion, proliferation and osteogenic differentiation of goat bone marrow-derived mesenchymal stem cells in vitro in a prolonged culture of 28 days. The cellular activities were significantly enhanced on HASi signifying the role of silica to stimulate osteoblast cells. The fabrication of such a ‘cell-ceramic construct using autologous MSCs’ is aimed for the transplantation to a large bone defect site in the goat femur model which still remains a formidable challenge in Orthopedic surgery.     

Design of novel three-phase PCL/TZ–HA biomaterials for use in bone regeneration applications

The design of bioactive scaffold materials able to guide cellular processes involved in new-tissue genesis is key determinant in bone tissue engineering. The aim of this study was the design and characterization of novel multi-phase biomaterials to be processed for the fabrication of 3D porous scaffolds able to provide a temporary biocompatible substrate for mesenchymal stem cells (MSCs) adhesion, proliferation and osteogenic differentiation. The biomaterials were prepared by blending poly(ε-caprolactone) (PCL) with thermoplastic zein (TZ), a thermoplastic material obtained by de novo thermoplasticization of zein. Furthermore, to bioactivate the scaffolds, microparticles of osteoconductive hydroxyapatite (HA) were dispersed within the organic phases. Results demonstrated that materials and formulations strongly affected the micro-structural properties and hydrophilicity of the scaffolds and, therefore, had a pivotal role in guiding cell/scaffold interaction. In particular, if compared to neat PCL, PCL–HA composite and PCL/TZ blend, the three-phase PCL/TZ–HA showed improved MSCs adhesion, proliferation and osteogenic differentiation capability, thus demonstrating potential for bone regeneration.     

Application of human amniotic mesenchymal cells as an allogeneic transplantation cell source in bone regenerative therapy

Autogenous mesenchymal stem cells (MSCs) have therapeutic applications in bone regenerative therapy due to their pluripotency. However, the ability of MSCs to proliferate and differentiate varies between donors. Furthermore, alternative sources of MSCs are required for patients with contraindications to autogenous cell therapy. The aim of this study was to evaluate the potential of mesenchymal cells from the human amniotic membrane (HAM) as a source of cells for allogeneic transplantation in bone regenerative therapy. Cells that retained a proliferative capacity of more than 50 population doubling level were distinguished from other HAM cells as HAMα cells and induced to osteogenic status—their in vivo osteogenesis was subsequently investigated in rats. It was found that HAMα cells were spindle shaped and were positive for MSC markers and negative for hematopoietic stem cell markers. Alkaline phosphatase activity and calcium deposition increased with osteogenic status of HAMα cells. The expression of osteocalcin mRNA was increased in HAMα cells cultured on calcium phosphate scaffolds. Moreover, xenografted HAMα cells remained viable and produced extracellular matrix for several weeks. Thus, this study suggests that human amniotic mesenchymal cells possess osteogenic differentiation potential and could be applied to allogeneic transplantation in bone regenerative therapy.     

Tethering of Gly-Arg-Gly-Asp-Ser-Pro-Lys Peptides on Mg-Doped Hydroxyapatite

Stem cell homing, namely the recruitment of mesenchymal stem cells (MSCs) to injured tissues, is highly effective for bone regeneration in vivo. In order to explore whether the incorporation of mimetic peptide sequences on magnesium-doped (Mg-doped) hydroxyapatite (HA) may regulate the homing of MSCs, and thus induce cell migration to a specific site, we covalently functionalized MgHA disks with two chemotactic/haptotactic factors: either the fibronectin fragment III₁-C human (FF III₁-C), or the peptide sequence Gly-Arg-Gly-Asp-Ser-Pro-Lys, a fibronectin analog that is able to bind to integrin transmembrane receptors. Preliminary biological evaluation of MSC viability, analyzed by 3-(4,5-dimethyl­thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test, suggested that stem cells migrate to the MgHA disks in response to the grafted haptotaxis stimuli.     

Mesenchymal stem cells combined with biphasic calcium phosphate ceramics promote bone regeneration

The reconstruction and repair of large bone defects, resulting from trauma, cancer or metabolic disorders, is a major clinical challenge in orthopaedics. Clinically available biological and synthetic grafts have clear limitations that necessitate the development of new graft materials and/or strategies. Human mesenchymal stem cells (MSCs), obtained from the adult bone marrow, are multipotent cells capable of differentiating into various mesenchymal tissues. Of particular interest is the ability of these cells to differentiate into osteoblasts, or bone-forming cells. At Osiris, we have extensively characterized MSCs and have demonstrated MSCs can induce bone repair when implanted in vivo in combination with a biphasic calcium phosphate, specifically hydroxyapatite/tricalcium phosphate. This article reviews previous and current studies utilizing mesenchymal stem cells and biphasic calcium phosphates in bone repair.