Osteogenic differentiation of adipose-derived stromal cells treated with GDF-5 cultured on a novel three-dimensional sintered microsphere matrix

BACKGROUND CONTEXT: It is well known that under the proper conditions multipotential bone marrow stromal cells are capable of osteogenic differentiation. Recently studies have demonstrated that an analogous subpopulation of cells exist within adipose tissue. Although early studies characterizing these adipose-derived stromal (ADS) cells in culture exist, investigations exploring the characteristics and viability of these cells cultured on a three-dimensional sintered microsphere matrix are absent. PURPOSE: To characterize and investigate the viability of ADS cells cultured on bioengineered three-dimensional sintered microsphere matrices (SMM). STUDY DESIGN: Basic science, laboratory study. PATIENT SAMPLE: Sixty SMM total. Six underwent examination by scanning electron microscopy, 18 for cellular viability, 18 for biochemical assay, and 18 for evaluation by gene expression. OUTCOME MEASURES: The SMM were examined under scanning electron microscopy to evaluate for adherence, migration, and proliferation at 7, 14, and 28 days. Cellular viability was assessed using colorimetric assay for mitochondrial dehydrogenases activity in viable cells (MTS [3-(4,5-dimethylthiazol-2-yl)5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay) at each corresponding time point. Osteoblastic differentiation was determined using biochemical assays for alkaline phosphatase activity and gene expression for alkaline phosphatase (ALP), osteocalcin (OC), and core binding factor alpha-1 (Cbfa1). METHODS: Multipotential ADS cells from adult Sprague Dawley rats were isolated and maintained in media. Sintered microsphere matrices of poly(lactide-co-glycolide) [85:15] were prepared using solvent evaporation technique followed by mechanical sieving and fabricated by heating in metal molds. ADS cells were then seeded on the SMM and cultured in media with growth and differentiation factor-5 (GDF-5). Treated samples and controls were evaluated at 7, 14, and 28 days. Statistical significance was set at p<.05. RESULTS: Multipotential ADS cells were capable of being isolated from adipose tissue. Scanning electron microscopy evaluation revealed cells adherent to the scaffold surface in a monolayer by 7 days. Cytoplasmic extensions were seen linking the cells on adjacent microspheres. Migration and proliferation resulting in extension of the cellular elements into the scaffold was apparent by 14 days. MTS confirmed cell viability within the scaffold throughout the 28-day study. Osteoblastic differentiation was confirmed using biochemical assays for alkaline phosphatase activity and gene expression for ALP, OC, and Cbfa1. CONCLUSIONS: This is the first study to investigate the fate of ADS seeded on a three-dimensional sintered microsphere matrix. The results of this study confirm that ADS cells, when treated with GDF-5, are not only capable of adhering to the bioengineered scaffold, but also remain viable and demonstrated the ability to migrate, proliferate, and subsequently undergo osteogenic differentiation under the conditions described. These early findings support the concept that ADS cells cultured on a SMM may serve as a viable alternative to more traditional methods of bone graft materials.     

Tissue-engineered provisional matrix as a novel approach to enhance diabetic wound healing

Inherent pathologies associated with diabetic wound microenvironment including increased proteolysis, inflammatory dysregulation, and impaired neovascularization prevent timely resolution of chronic diabetic ulcers. It is hypothesized that augmentation of local wound microenvironment with a stable provisional matrix formed by proteolysis-resistant angiogenic peptide nanofibers (NFs) will create permissive environment for attenuated inflammation, enhanced neovascularization, and improved diabetic wound healing. Using murine excisional wound healing models, full-thickness dorsal skin wounds were treated with either NFs or control solutions (phosphate buffered saline; hyaluronic acid) and analyzed for morphology, inflammatory response, neovascularization, and biomechanical properties. NF treatment of diabetic wounds stimulated formation of a robust pro-angiogenic in situ tissue-engineered provisional matrix leading to a significant decrease in wound inflammatory cell infiltration and proinflammatory interleukin-6 levels, a significant increase in endothelial and endothelial progenitor cell infiltration, vascular endothelial growth factor levels, and neovascularization (day 7), as well as improved wound morphology, accelerated wound closure, and significantly stronger repair tissue (day 28). These results suggest that appropriate design of provisional matrix may compensate for some of the complex disruptions in diabetic wound microenvironment and provide missing cues to cells and direct in situ responses toward improved healing, which is promising for future development of new therapies for diabetic ulcers.     

PRP modulates expression of bone matrix proteins in vivo without long-term effects on bone formation

This experimental study (domestic pig) examined the bone formation after filling defined defects of the frontal skull with autogenous bone or a deproteinized bovine bone matrix (DBBM) in combination with platelet-rich plasma (PRP). Six groups, both materials with and without PRP in two different concentrations (4.1x and 6.5x referring to untreated whole blood) were evaluated at 2, 4, 12, and 26 weeks by means of immunohistochemical staining for different bone matrix proteins, microradiography, light microscopy and polychromatic fluorescence labeling. The sequential expression of bone matrix proteins reflected the specific roles these proteins fulfil in the mineralization of hard tissue. Collagen I expression at 2 weeks was enhanced in all autogenous bone groups. No specific modification of the collagen I expression was found after use of DBBM with or without PRP. Osteopontin and especially osteonectin showed a remarkable enhancement at 4 weeks in nearly all autogenous bone and DBBM groups. These increased levels closely resembled the mineralization content evaluated by microradiography at that time. For the three autogenous bone groups, an expression peak for osteocalcin was demonstrated at 12 weeks, further reflecting the way of de novo bone formation. The microradiographic evaluation demonstrated a statistically significant enhancement in bone regeneration by PRP only after use of autogenous bone plus PRP at 2 weeks (P = 0.002). After 4 weeks, mineralization values after use of autogenous bone were significantly lower if PRP was added to the autogenous bone (P = 0.002). No long-term effects of the PRP administration were found in the mineralization process. In all DBBM groups, bone formation remained unchanged, confirming the lack of any osteoinductive capacity of PRP. PRP modulated the expression of bone matrix proteins in this experimental setting. However, an enhancement of bone formation was demonstrated only at 2 weeks after application of the higher PRP concentration in combination with autogenous bone. In conjunction with an anorganic bovine bone no effects of PRP on defect mineralization were discovered, demonstrating the lack of osteoinductive capacity in PRP as well as in DBBM.     

Tissue-engineered colon exhibits function in vivo

BACKGROUND: Postcolectomy morbidities include important changes in enterohepatic circulation, stool microbiology, and absorption. The surgical substitution of an ileal pouch for the absent colon also has a number of serious complications. We report in vivo colon replacement by tissue-engineered colon (TEC) in lieu of an ileal pouch. METHODS: End-ileostomies were created in 22 male Lewis rats. In 11 animals, side-to-side ileum-TEC anastomosis was performed 1 cm from the stoma. This group was compared with end-ileostomy alone. Serial weights were measured, and animals were harvested sequentially for assessment of histologic signs of pouchitis. Transit times, stool dry and wet weights, and serum and stool colon function markers were collected. RESULTS: Animals survived 41 days. Weight loss was more than 1.5 times greater in the end-ileostomy alone group compared with the ileum-TEC group. Transit times were significantly longer in the ileum-TEC group than the end-ileostomy alone group, with lower stool moisture content and higher total serum bile acids. Animals without TEC had statistically significant hyponatremia, elevated serum urea nitrogen, and lower stool short chain fatty acids (13.5 micromol/kg vs 84.2) with an abnormal distribution. CONCLUSIONS: TEC successfully recapitulates some major physiologic functions of native large intestine in vivo.     

完全脱蛋白骨复合rhBMP2构建人工骨及其体内异位成骨

目的: 探讨异种完全脱蛋白骨复合基因重组骨形态发生蛋白(rhBMP2) 在体外构建人工骨的可行性,并对其体内异位成骨进行评估。方法: 体外将兔骨髓基质细胞进行成骨诱导, 然后接种复合rhBMP2的小牛完全脱蛋白骨, 构建组织工程骨, 植入自体大腿肌袋内, 术后4、8周处死动物分别进行常规组织学检查、碱性磷酸酶活性测定、折弯力学测试, 观测这种组织工程骨在体内的异位成骨作用。结果: 这种方法构建和组织工程骨在体内异位成骨效应显著且抗折能力较强。结论: 异种完全脱蛋白骨复合rhBMP2可作为骨组织工程支架材料, 这种组织工程骨在体内的成骨能力随植入时间的延长而增加。     

Tissue-engineered bone constructed in a bioreactor for repairing critical-sized bone defects in sheep

Purpose

Repair of bone defects, particularly critical-sized bone defects, is a considerable challenge in orthopaedics. Tissue-engineered bones provide an effective approach. However, previous studies mainly focused on the repair of bone defects in small animals. For better clinical application, repairing critical-sized bone defects in large animals must be studied. This study investigated the effect of a tissue-engineered bone for repairing critical-sized bone defect in sheep.

Methods

A tissue-engineered bone was constructed by culturing bone marrow mesenchymal-stem-cell-derived osteoblast cells seeded in a porous β-tricalcium phosphate ceramic (β-TCP) scaffold in a perfusion bioreactor. A critical-sized bone defect in sheep was repaired with the tissue-engineered bone. At the eighth and 16th week after the implantation of the tissue-engineered bone, X-ray examination and histological analysis were performed to evaluate the defect. The bone defect with only the β-TCP scaffold served as the control.

Result

X-ray showed that the bone defect was successfully repaired 16 weeks after implantation of the tissue-engineered bone; histological sections showed that a sufficient volume of new bones formed in β-TCP 16 weeks after implantation. Eight and 16 weeks after implantation, the volume of new bones that formed in the tissue-engineered bone group was more than that in the β-TCP scaffold group (P?Conclusion Tissue-engineered bone improved osteogenesis in vivo and enhanced the ability to repair critical-sized bone defects in large animals.     

Posterolateral lumbar spine fusion using a novel demineralized bone matrix: a controlled case pilot study

Introduction

Intertransverse posterolateral fusion along with instrumentation is a common technique used for spinal fusion. Iliac crest bone graft (ICBG) offers good fusion success rates with a low risk for disease transmission but is, however, linked with certain morbidity. In an effort to eliminate or reduce the amount of iliac graft needed, bone substitutes including demineralized bone matrix (DBM) have been developed. This study evaluates a novel DBM (Accell Connexus^®) used in one or two-level instrumented posterolateral lumbar fusion.

Materials and methods

A total of 59 consecutive patients were studied as two groups. Group 1 consisted of 33 patients having Accell Connexus^® used to augment either ICBG or local decompression material. Group 2 consisted of 26 consecutive patients, operated prior to the introduction of this novel DBM, having either ICBG alone or local decompression material. Fusion was assessed by two independent observers, blinded to graft material, using standardized criteria found in the literature. All adverse events were recorded prospectively.

Results

The results show no statistically significant differences between the two groups in fusion rates, complications, surgery duration, ODI, or pain on VAS. Logistical regression showed no relation between fusion and age, smoking status or comorbidities. Furthermore, no adverse events related to the use of the novel DBM were observed.

Conclusion

The results from this study demonstrate that the novel DBM presented performs equally as well as that of autologous bone, be it either ICBG or a local decompression material, and can therefore be used as a graft extender.

     

Rabbit bone matrix induces bone formation in the athymic rat

Rabbit and rat bone matrix were implanted in athymic rat muscle, and the bone yield was measured as total calcium content after 4, 6, and 8 weeks. Matrix from both species induced equal amounts of new bone in the athymic rat. In rabbit and normal rat, the xenogenic matrix induced little or no bone formation. Thus, in the case of rabbit and rat, bone induction is species specific due to immunogenic mechanisms. The athymic rat can be used to measure inductive properties of bone matrix from different species.     

Tissue-engineered cartilage using fibrin/hyaluronan composite gel and its in vivo implantation

The importance of scaffold biomaterials has been emphasized for in vitro culture of tissue-engineered cartilage in a three-dimensional (3D) environment. In this study, we examined the feasibility of fibrin glue, mixed with hyaluronic acid (HA) as a composite scaffold. Fibrin glue has been a useful cell delivery matrix for cartilage tissue engineering and HA is a key component of normal articular cartilage. Our hypothesis is that compared to fibrin itself, a fibrin/HA composite can have significantly enhanced properties, due mainly to the added benefits of HA in the matrix. Pieces of cartilage were isolated from rabbit knees and the chondrocytes were harvested through enzymatic digestion. Both fibrin and fibrin/HA composite were prepared and subsequently implanted in nude mice (n = 9, each group) for 1, 2, and 4 weeks, respectively. The retrieved specimens were then analyzed and the results were compared. Cartilage-like tissue formation was detected earlier with fibrin/HA specimens. They produced significantly higher amounts of the extracellular matrix (ECM) molecules, GAG, and collagen at each time point than those in fibrin. Interestingly, the fibrin/HA composite was also competent in maintaining its initial size. Histology--Safranin O/fast green and Alcian blue--of the retrieved specimens found more intense, uniform staining in the fibrin/HA composites. Analysis of the gene expression of the ECM molecules also confirmed the benefits of the composite with added HA in the maintenance of phenotypic stability. The present study suggests that fibrin/HA composite may serve as a dependable cell delivery vehicle as well as a structural basis for tissue-engineered cartilage.     

Rabbit bone matrix induces bone formation in the athymic rat

Rabbit and rat bone matrix were implanted in athymic rat muscle, and the bone yield was measured as total calcium content after 4, 6, and 8 weeks. Matrix from both species induced equal amounts of new bone in the athymic rat. In rabbit and normal rat, the xenogenic matrix induced little or no bone formation. Thus, in the case of rabbit and rat, bone induction is species specific due to immunogenic mechanisms. The athymic rat can be used to measure inductive properties of bone matrix from different species.     

Longitudinal in vivo imaging of bone formation and resorption using fluorescence molecular tomography

Bone research often focuses on anatomical imaging of the bone microstructure, but in order to gain better understanding in how bone remodeling is modulated through interventions also bone formation and resorption processes should be investigated. With this in mind, the purpose of this study was to establish a longitudinal in vivo imaging approach of bone formation and resorption using fluorescence molecular tomography (FMT). In this study the reproducibility, accuracy and sensitivity of FMT for bone imaging were assessed by performing longitudinal measurements with FMT and comparing it to in vivo micro-computed tomography on a set of control mice, and mice in which load-adaptation was induced in the sixth caudal vertebra. The precision error for FMT measurements, expressed as coefficient of variation, was smaller than 16%, indicating acceptable reproducibility. A correlation was found between bone resorption measured with FMT and bone resorption rate measured with in vivo micro-computed tomography only over the first 14 days (R = 0.81, p < 0.01), but not between bone formation measured with FMT and bone formation rate measured with in vivo micro-CT. Bone formation measured by FMT was 89–109% greater (p < 0.05) for mice subjected to mechanical loading than control mice. Bone resorption was 5–8% lower, but did not reach a significant difference between groups, indicating moderate sensitivity for FMT. In conclusion, in vivo FMT in mouse tail bones is feasible but needs to be optimized for monitoring load adaptation in living mice.     

掺锶硫酸钙及DBM复合材料骨修复能力评价

[目的]制备一种掺锶硫酸钙与DBM复合的骨修复材料,为临床提供一种成骨活性优良的骨修复材料。[方法]制备掺锶硫酸钙与DBM复合的活性骨修复材料,其中锶元素的量为钙含量的6%,掺锶硫酸钙与DBM按体积比1∶1复合。观察兔股骨髁骨缺损模型修复效果,利用X线片观察缺损区域的新骨形成情况,采用MicroCT评价局部的骨量和骨质变化,对组织切片予以Van Gieson(VG)复合染色法染色进行组织形态学观察,荧光双标观测计算成骨速率。[结果]术后动物无死亡,所有兔子切口无渗血或出现分泌物状况,术后活动正常。从MicroCT的BV/TV结果看出实验组在12周时缺损区域已经恢复;掺锶CaSO_4复合材料组缺损部位已经基本修复,CaSO_4组未完全修复。VG组织学观察显示各组均有新生骨小梁生成,荧光双标观测得到的矿化沉积率显示复合材料组新骨生长突出,矿化沉积率为(2.31±0.13)μm/d。[结论]通过MicroCT、VG组织学染色以及荧光双标观测对兔子股骨髁缺损修复效果进行评价,发现复合材料具有较好的骨缺损修复能力。     

Accelerating bone formation and earlier healing after using demineralized bone matrix for limb lengthening in rabbits.

We studied the effects of grafting with demineralized bone matrix during lengthening of the tibia in young Japanese White rabbits. The demineralized bone matrix was made from frozen cortical bone harvested from other rabbits. A 5-mm gap was created in the tibial diaphysis by a subperiosteal osteotomy; a maximum of 20 mm (2-3 mm/day) of tibial lengthening was reached in a week with use of an external fixator. The control group of 20 rabbits did not receive an implant; the group of 21 experimental rabbits received an implant of demineralized bone matrix in the surgical gap. The control group failed to demonstrate radiographic callus 5 weeks after surgery, and nonunion was persistent after 1 year. In the group with demineralized bone matrix, new radiodensity was demonstrated within the lengthening gap at 3 weeks, with a gradual increase in bone density to 85% that of the intact tibia after 12 weeks. Bone union was seen within 1 year for all experimental animals for whom the external fixator was removed 8 weeks after the procedure. These bones showed normal bone structure histologically. The lengthening was carried out at a rapid distraction rate of 2-3 mm/day; therefore, this method allows for satisfactory bone formation at a faster rate than normal.     

Epithelial cell lines that induce bone formation in vivo produce alkaline phosphatase-enriched matrix vesicles in culture.

Hypertrophic chondrocytes and osteoblasts produce alkaline phosphatase (ALPase)-enriched matrix vesicles in vivo and in vitro and, along with certain epithelial cell lines and osteoblast precursors, induce bone when implanted in mesenchymal tissues. This study examined whether ALPase-enriched matrix vesicle production in vitro was a general property of cells that induce bone in vivo. Epithelial cell lines FL, WISH, and OK 16; connective tissue cell lines HEPM 1 and HEPM 2; neonatal rat muscle cells; rat costochondral chondrocytes; and human fibroblasts were implanted intramuscularly into nude mice. The FL and WISH cells produced tumors and induced large islands of bone with focal areas of cartilage immediately adjacent to the tumors. The chondrocytes formed cartilage nodules but did not induce bone, indicating that the ability of the cells to form a solid mass was not an a priori requirement for bone formation. No other cell type produced tumors or nodules or induced bone formation, although connective tissue cells have been shown to induce chondrogenesis in vitro and osteogenesis in vivo. Only matrix vesicles from normal chondrocytes, FL, WISH, and OK16 cultures exhibited enriched ALPase-specific activity. Matrix vesicles from FL and WISH cultures exhibited ALPase specific activities similar to those isolated from osteoblast or chondrocyte cultures. These data suggest that the ability to produce ALPase-enriched matrix vesicles in culture may be associated with the ability of cells to induce bone or cartilage in vivo.     

Tissue-engineered cartilage and bone using stem cells from human infrapatellar fat pads

Multipotential processed lipoaspirate (PLA) cells extracted from five human infrapatellar fat pads and embedded into fibrin glue nodules, were induced into the chondrogenic phenotype using chondrogenic media. The remaining cells were placed in osteogenic media and were transfected with an adenovirus carrying the cDNA for bone morphogenetic protein-2 (BMP-2). We evaluated the tissue-engineered cartilage and bone using in vitro techniques and by placing cells into the hind legs of five severe combined immunodeficient mice. After six weeks, radiological and histological analysis indicated that the PLA cells induced into the chondrogenic phenotype had the histological appearance of hyaline cartilage. Cells transfected with the BMP-2 gene media produced abundant bone, which was beginning to establish a marrow cavity. Tissue-engineered cartilage and bone from infrapatellar fat pads may prove to be useful for the treatment of osteochondral defects.     

An experimental approach to spinal fusion using sintered bovine bone in a pig model

Sintered bovine bone is a biomaterial based on calcium phosphate, an organized crystal of bone mineral that possesses a natural trabecular structure. The authors considered whether sintered bovine bone can integrate with recipient bone and adjust to the strength of recipient bone for anterior spinal fusion in an animal model. Either autologous iliac bone or sintered bovine bone was implanted for a spinal body fusion. Some pigs underwent exposure of the spinal bodies without implantation. Based on radiographic evaluation, manual palpation, biomechanical testing, and histologic examination, spinal fusion with sintered bovine bone resulted in a composition and structure similar to that of the autograft (or of no implantation). The sintered bovine bone with its moderate strength tended to adjust to the bone stiffness of the host bone in the specimens as new bone grew.     

Induction of ectopic bone formation by using human periosteal cells in combination with a novel scaffold technology

Due to their osteogenic germination potential, periosteum-derived osteoprogenitor cells are a potential source for tissue engineering a bone graft that could be used to regenerate skeletal defects. In this study we evaluated if ectopic bone formation could be induced by a construct made of human periosteal cells and a novel scaffold architecture whose mechanical properties are in the range of cancellous bone. Biopsies from human calvarial periosteum were harvested and cells were isolated from the inner cambial layer. Fifty thousand periosteal cells were seeded into the scaffolds measuring 6 x 6 x 2 mm. The cell-scaffold constructs were cultured for a period of 3 weeks prior to implantation into balb C nude mice. Mice were sacrificed and implants were analyzed 6 and 17 weeks postoperatively. Immunohistochemical analysis confirmed the osteoblastic phenotype of the seeded cells. Formation of focal adhesions and stress fibers could be observed in both scaffold architectures. Three-dimensional cell proliferation was observed after 2 weeks of culturing with centripetal growth pattern inside the pore network. The deposition of calcified extracellular matrix was observed after 3 weeks of culturing. In vivo, endochondral bone formation with osteoid production was detectable via von Kossa and Osteocalcin staining after 6 and 17 weeks. Histology and SEM revealed that the entire scaffold/bone grafts were penetrated by a vascular network. This study showed the potential of bone tissue engineering by using human periosteal cells in combination with a novel scaffold technology.     

Cementum matrix formation in vivo by cultured dental follicle cells

Dental follicle is the fibrous tissue that surrounds the developing tooth germ, and it is believed to contain progenitors for cementoblasts, periodontal ligament cells, and osteoblasts. In this study, we report the presence of cementoblast progenitors in cultures of bovine dental follicle cells and demonstrate their differentiation capacity. Bovine dental follicle cells (BDFC) obtained from tooth germs by collagenase digestion were compared with bovine alveolar bone osteoblasts (BAOB) and bovine periodontal ligament cells (BPDL) in vitro and in vivo. In culture, BDFC exhibited low levels of alkaline phosphatase activity and expressed mRNA for osteopontin (OP) and type I collagen (COLI), as well as low levels of osteocalcin (OC) mRNA. In contrast, cultured BAOB exhibited high alkaline phosphatase activity levels and expressed mRNA for OC, OP, COLI, and bone sialoprotein (BSP). To elucidate the differentiation capacity of BDFC in vivo, cells were transplanted into severe combined immunodeficiency (SCID) mice and analyzed after 4 weeks. Transplanted BDFC formed fibrous tissue and cementum-like matrix, which stained positive for anti-cementum attachment protein (CAP) monoclonal antibody (3G9), and expressed mRNA for OC, OP, COLI, and BSP. On the other hand, transplanted BAOB formed bone-like matrix, but were negative for anti-CAP monoclonal antibody. The BPDL transplants formed fibrous tissue that contained a few cells expressing CAP. These results indicate that cementoblast progenitors are present in BDFC, which can provide a useful model for investigating the molecular mechanisms of cementogenesis.