Sphingosine-1-phosphate mediates proliferation maintaining the multipotency of human adult bone marrow and adipose tissue-derived stem cells

High renewal and maintenance of multipotency of human adult stem cells (hSCs), are a prerequisite for experimental analysis as well as for potential clinical usages. The most widely used strategy for hSC culture and proliferation is using serum. However, serum is poorly defined and has a considerable degree of inter-batch variation, which makes it difficult for large-scale mesenchymal stem cells (MSCs) expansion in homogeneous culture conditions. Moreover, it is often observed that cells grown in serum-containing media spontaneously differentiate into unknown and/or undesired phenotypes. Another way of maintaining hSC development is using cytokines and/or tissue-specific growth factors; this is a very expensive approach and can lead to early unwanted differentiation. In order to circumvent these issues, we investigated the role of sphingosine-1-phosphate (S1P), in the growth and multipotency maintenance of human bone marrow and adipose tissue-derived MSCs. We show that S1P induces growth, and in combination with reduced serum, or with the growth factors FGF and platelet-derived growth factor-AB, S1P has an enhancing effect on growth. We also show that the MSCs cultured in S1P-supplemented media are able to maintain their differentiation potential for at least as long as that for cells grown in the usual serum-containing media. This is shown by the ability of cells grown in S1P-containing media to be able to undergo osteogenic as well as adipogenic differentiation. This is of interest, since S1P is a relatively inexpensive natural product, which can be obtained in homogeneous high-purity batches: this will minimize costs and potentially reduce the unwanted side effects observed with serum. Taken together, S1P is able to induce proliferation while maintaining the multipotency of different human stem cells, suggesting a potential for S1P in developing serum-free or serum-reduced defined medium for adult stem cell cultures.

Typical accuracy and quality control of a process for creating CT-Based virtual bone models

A pragmatic method for assessing the accuracy and precision of a given processing pipeline required for converting computed tomography (CT) image data of bones into representative three dimensional (3D) models of bone shapes is proposed. The method is based on coprocessing a control object with known geometry which enables the assessment of the quality of resulting 3D models. At three stages of the conversion process, distance measurements were obtained and statistically evaluated. For this study, 31 CT datasets were processed. The final 3D model of the control object contained an average deviation from reference values of −1.07±0.52 mm standard deviation (SD) for edge distances and −0.647±0.43 mm SD for parallel side distances of the control object. Coprocessing a reference object enables the assessment of the accuracy and precision of a given processing pipeline for creating CTbased 3D bone models and is suitable for detecting most systematic or human errors when processing a CT-scan. Typical errors have about the same size as the scan resolution.

The effect of friction on indenter force and pile-up in numerical simulations of bone nanoindentation

Nanoindentation is a useful technique for probing the mechanical properties of bone, and finite element (FE) modeling of the indentation allows inverse determination of elasto-plastic constitutive properties. However, FE simulations to date have assumed frictionless contact between indenter and bone. The aim of this study was to explore the effect of friction in simulations of bone nanoindentation. Two dimensional axisymmetric FE simulations were performed using a spheroconical indenter of tip radius 0.6m and angle 90°. The coefficient of friction between indenter and bone was varied between 0.0 (frictionless) and 0.3. Isotropic linear elasticity was used in all simulations, with bone elastic modulus E=13.56GPa and Poisson’s ratio =0.3. Plasticity was incorporated using both Drucker-Prager and von Mises yield surfaces. Friction had a modest effect on the predicted force-indentation curve for both von Mises and Drucker-Prager plasticity, reducing maximum indenter displacement by 10% and 20% respectively as friction coefficient was increased from zero to 0.3 (at a maximum indenter force of 5mN). However, friction has a much greater effect on predicted pile-up after indentation, reducing predicted pile-up from 0.27m to 0.11m with a von Mises model, and from 0.09m to 0.02m with Drucker-Prager plasticity. We conclude that it is important to include friction in nanoindentation simulations of bone.

Repair of calvarial defects with customized tissue-engineered bone grafts - I. Evaluation of osteogenesis in a three-dimensional culture system

Bone generation by autogenous cell transplantation in combination with a biodegradable scaffold is one of the most promising techniques being developed in craniofacial surgery. The objective of this combined in vitro and in vivo study was to evaluate the morphology and osteogenic differentiation of bone marrow derived mesenchymal progenitor cells and calvarial osteoblasts in a two-dimensional (2-D) and three-dimensional (3-D) culture environment (Part I of this study) and their potential in combination with a biodegradable scaffold to reconstruct critical-size calvarial defects in an autologous animal model [Part II of this study; see Schantz, J.T., et al. Tissue Eng. 2003;9(Suppl. 1):S-127-S-139; this issue]. New Zealand White rabbits were used to isolate osteoblasts from calvarial bone chips and bone marrow stromal cells from iliac crest bone marrow aspirates. Multilineage differentiation potential was evaluated in a 2-D culture setting. After amplification, the cells were seeded within a fibrin matrix into a 3-D polycaprolactone (PCL) scaffold system. The constructs were cultured for up to 3 weeks in vitro and assayed for cell attachment and proliferation using phase-contrast light, confocal laser, and scanning electron microscopy and the MTS cell metabolic assay. Osteogenic differentiation was analyzed by determining the expression of alkaline phosphatase (ALP) and osteocalcin. The bone marrow-derived progenitor cells demonstrated the potential to be induced to the osteogenic, adipogenic, and chondrogenic pathways. In a 3-D environment, cell-seeded PCL scaffolds evaluated by confocal laser microscopy revealed continuous cell proliferation and homogeneous cell distribution within the PCL scaffolds. On osteogenic induction mesenchymal progenitor cells (12 U/L) produce significantly higher (p < 0.05) ALP activity than do osteoblasts (2 U/L); however, no significant differences were found in osteocalcin expression. In conclusion, this study showed that the combination of a mechanically stable synthetic framework (PCL scaffolds) and a biomimetic hydrogel (fibrin glue) provides a potential matrix for bone tissue-engineering applications. Comparison of osteogenic differentiation between the two mesenchymal cell sources revealed a similar pattern.

Application of micro CT and computation modeling in bone tissue engineering

Computer aided technologies, medical imaging, and rapid prototyping has created new possibilities in biomedical engineering. The systematic variation of scaffold architecture as well as the mineralization inside a scaffold/bone construct can be studied using computer imaging technology and CAD/CAM and micro computed tomography (CT). In this paper, the potential of combining these technologies has been exploited in the study of scaffolds and osteochondral repair. Porosity, surface area per unit volume and the degree of interconnectivity were evaluated through imaging and computer aided manipulation of the scaffold scan data. For the osteochondral model, the spatial distribution and the degree of bone regeneration were evaluated. In this study the versatility of two softwares Mimics (Materialize), CTan and 3D realistic visualization (Skyscan) were assessed, too.

Clonal characterization of bone marrow derived stem cells and their application for bone regeneration

Tissue engineering allows the design of functionally active cells within supportive bio-scaffolds to promote the development of new tissues such as cartilage and bone for the restoration of pathologically altered tissues. However, all bone tissue engineering applications are limited by a shortage of stem cells. The adult bone marrow stroma contains a subset of nonhematopoietic cells referred to as bone marrow mesenchymal stem cells (BMSCs). BMSCs are of interest because they are easily isolated from a small aspirate of bone marrow and readily generate single- cell-derived colonies. These cells have the capacity to undergo extensive replication in an undifferentiated state ex vivo. In addition, BMSCs have the potential to develop either in vitro or in vivo into distinct mesenchymal tissues, including bone, cartilage, fat, tendon, muscle, and marrow stroma. Thus, BMSCs are an attractive cell source for tissue engineering approaches. However, BMSCs are not homo- geneous and the quantity of stem cells decreases in the bone marrow in aged population. A sequential loss of lineage differentiation potential has been found in the mixed culture of bone marrow stromal cells due to a heterogenous popu- lation. Therefore, a number of studies have proposed that homogenous bone marrow stem cells can be generated from clonal culture of bone marrow cells and that BMSC clones have the greatest potential for the application of bone regeneration in vivo

Is it necessary to re-fuse a non-union of a hallux metatarsophalangeal joint arthrodesis?

BACKGROUND: The standard treatment for a non-union of the hallux metatarsophalangeal joint fusion has been to revise the fusion. Revision fusion is technically more demanding, often involving bone grafting, more substantial fixation and prolonged period of immobilization postoperatively. We present data to suggest that removal of hardware and debridement alone is an alternative treatment option. ---------- MATERIALS AND METHODS: A case note review identified patients with a symptomatic non-union after hallux metatarsophalangeal joint (MTPJ) fusion. It is our practice to offer these patients revision fusion or removal of hardware and debridement. For the seven patients that chose hardware removal and were left with a pseudarthrosis, a matched control group was selected from patients who had had successful fusions. Three outcome scores were used. Hallux valgus and dorsiflexion angles were recorded.---------- RESULTS: One hundred thirty-nine hallux MTPJ arthrodeses were carried out. Fourteen non-unions were identified. The rate of non-union in males and following previous hallux MTPJ surgery was 19% and 24%, respectively. In females undergoing a primary MTPJ fusion, the rate was 2.4%. Twelve non-union patients were reviewed at 27 months (mean). Eleven patients had elected to undergo removal of hardware and debridement. Four patients with pseudarthrosis were unhappy with the results and proceeded to either revision fusion or MTPJ replacement. Seven non-union patients, who had removal of hardware alone, had outcome scores marginally worse compared to those with successful fusions.---------- CONCLUSION: Removal of hardware alone is a reasonable option to offer as a relatively minor procedure following a failed arthrodesis of the first MTPJ. This must be accepted on the proviso that in this study four out of 11 (36%) patients proceeded to a revision first MTPJ fusion or first MTPJ replacement. We also found that the rate of non-union in primary first MTPJ fusion was significantly higher in males and those patients who had undergone previous surgery.

An alginate-based hybrid system for growth factor delivery in the functional repair of large bone defects

The treatment of challenging fractures and large osseous defects presents a formidable problem for orthopaedic surgeons. Tissue engineering/regenerative medicine approaches seek to solve this problem by delivering osteogenic signals within scaffolding biomaterials. In this study, we introduce a hybrid growth factor delivery system that consists of an electrospun nanofiber mesh tube for guiding bone regeneration combined with peptide-modified alginate hydrogel injected inside the tube for sustained growth factor release. We tested the ability of this system to deliver recombinant bone morphogenetic protein-2 (rhBMP-2) for the repair of critically-sized segmental bone defects in a rat model. Longitudinal [mu]-CT analysis and torsional testing provided quantitative assessment of bone regeneration. Our results indicate that the hybrid delivery system resulted in consistent bony bridging of the challenging bone defects. However, in the absence of rhBMP-2, the use of nanofiber mesh tube and alginate did not result in substantial bone formation. Perforations in the nanofiber mesh accelerated the rhBMP-2 mediated bone repair, and resulted in functional restoration of the regenerated bone. [mu]-CT based angiography indicated that perforations did not significantly affect the revascularization of defects, suggesting that some other interaction with the tissue surrounding the defect such as improved infiltration of osteoprogenitor cells contributed to the observed differences in repair. Overall, our results indicate that the hybrid alginate/nanofiber mesh system is a promising growth factor delivery strategy for the repair of challenging bone injuries.