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Bone defects caused by various causes remain a major problem in orthopedic clinics. A number of different treatments have been developed and proposed, but until now, none has proven to be completely satisfactory. For 26 patients with bone defects but limited autologous bone source or allogeneic bone graft failure, we used individual tissue-engineered bones (iTEBs) for repairing, which were constructed by autologous bone marrow mesenchymal stem cells and allogenic decalcified bone matrix (DBM) scaffolds. The clinical outcomes, including efficacy and safety, were evaluated by radiological examinations, postoperative function recovery score and laboratory tests. Twenty-six patients, including 18 men and 8 women, were followed up for an average of 10 years to analyze the long-term outcome. The mean healing time for patients with lacunar bone defects was 3.87 ± 2.01 months (range, 2-9 months) and that for structural bone defects was longer than 12 months. The Musculoskeletal Tumor Society functional evaluation system and the Barthel Index scores were significantly improved during the long-term follow-up. The white blood cell, erythrocyte sedimentation rate, C reactive protein, complement, immunoglobulins, and liver and renal functions were not significantly affected by bone grafting. One patient with bone cyst relapsed at 3 years postoperatively and achieved bone healing after re-transplantation. No tumorigenesis, tumor metastasis, or blood transmissible disease was found in the whole process. The results demonstrated that iTEBs were effective and safe for repairing bone defects in the long period, especially for those with lacunar bone defects and limited autograft source. Impact statement Currently, controversies exist about the long-term safety and effectiveness of the clinical application of tissue-engineered bones (TEBs) due to potential tumorigenesis, immune rejection, disease transmission, and others. In this study, we show that individual TEBs constructed by autologous MSCs and allogenic decalcified bone matrix are reliable for repairing bone defects in regard to its long-term safety and effectiveness. Our study provides experience and basis about the clinical application of TEBs in the treatment of bone defects.

Patient-specific individual tissue-engineered bones (iTEBs) have been recognized as a promising strategy for treating large bone defects. However, current construction protocols of iTEBs vary between lots and lack standardization and quality control, hampering further research and application. This study was aimed to detail a standardized constructing protocol for iTEBs, which can be used for both clinical and experimental purposes. The procedure was designed and described as follows: scaffold preparation, cell isolation and culture, and fabrication of iTEBs. Manipulation and caution points in each section were detailed. A series of scales on the quality control and safety monitoring was developed. The effectiveness and safety of iTEBs were evaluated. Eventually, the preparing portion, from cell culture to scaffold treatment, usually required 21 days. Generally, the fabrication section took 5 days. The main advantage of this protocol was that each step was standardized and quality controlling and safety monitoring were performed throughout the process to ensure the homogeneity, reliability, and safety. The resulting iTEBs were effective and applicable to both clinical and experimental purposes. Thus, we have established a refined and standardized protocol detailing the construction process of patient-specific iTEBs that comply with strict quality control and safety criteria. This protocol is relatively easy for graduate students or staff working in the field of bone tissue engineering to implement.

To date, various types of cells for seeding regenerative scaffolds have been used for bone tissue engineering. Among seed cells, the mesenchymal stem cells derived from human umbilical cord Wharton's jelly (hUCMSCs) represent a promising candidate and hold potential for bone tissue engineering due to the the lack of ethical controversies, accessibility, sourced by non-invasive procedures for donors, a reduced risk of contamination, osteogenic differentiation capacities, and higher immunomodulatory capacity. However, the current culture methods are somewhat complicated and inefficient and often fail to make the best use of the umbilical cord (UC) tissues. Moreover, these culture processes cannot be performed on a large scale and under strict quality control. As a result, only a small quantity of cells can be harvested using the current culture methods. To solve these problems, we designed and evaluated an UC Wharton's jelly repeated culture device. Using this device, hUCMSCs were obtained from the repeated cultures and their quantities and biological characteristics were compared. We found that using our culture device, which retained all tissue blocks on the bottom of the dish, the total number of obtained cells increased 15-20 times, and the time required for the primary passage was reduced. Moreover, cells harvested from the repeated cultures exhibited no significant difference in their immunophenotype, potential for multilineage differentiation, or proliferative, osteoinductive capacities, and final osteogenesis. The application of the repeated culture frame (RCF) not only made full use of the Wharton's jelly but also simplified and specified the culture process, and thus, the culture efficiency was significantly improved. In summary, abundant hUCMSCs of dependable quality can be acquired using the RCF.

The purpose of this study was to evaluate the effect of different frequencies of pulsed electromagnetic fields on the osteogenic differentiation of human mesenchymal stem cells. Third-generation human mesenchymal stem cells were irradiated with different frequencies of pulsed electromagnetic fields, including 5, 25, 50, 75, 100, and 150 Hz, with a field intensity of 1.1 mT, for 30 minutes per day for 21 days. Changes in human mesenchymal stem cell morphology were observed using phase contrast microscopy. Alkaline phosphatase activity and osteocalcin expression were also determined to evaluate human mesenchymal stem cell osteogenic differentiation.Different effects were observed on human mesenchymal stem cell osteoblast induction following exposure to different pulsed electromagnetic field frequencies. Levels of human mesenchymal stem cell differentiation increased when the pulsed electromagnetic field frequency was increased from 5 hz to 50 hz, but the effect was weaker when the pulsed electromagnetic field frequency was increased from 50 Hz to 150 hz. The most significant effect on human mesenchymal stem cell differentiation was observed at of 50 hz.The results of the current study show that pulsed electromagnetic field frequency is an important factor with regard to the induction of human mesenchymal stem cell differentiation. Furthermore, a pulsed electromagnetic field frequency of 50 Hz was the most effective at inducing human mesenchymal stem cell osteoblast differentiation in vitro., (Copyright 2012, SLACK Incorporated.)

Objective: The combined application of green fluorescent protein (GFP) and confocal laser scanning microscope three-dimensional reconstruction (CLSM-3DR) were used to monitor the construction and in vivo transplantation of tissue engineered bone (TEB), to provide for technology in selection of scaffolds and three-dimensional constructional methods., Methods: After bone marrow mesenchymal stem cells (BMSCs) were isolated from a 2-year-old green goat by a combination method of density gradient centrifugation and adherent culture, and the expressions of CD29, CD60L, CD45, and CD44 in BMSCs were detected by flow cytometry. Plasmid of pLEGFP-N1 was amplified, digested by enzymes (Hind III, BamH I, Sal I, and Bgl II), and identified. Transfection of pLEGFP-N1 into PT67 cells was performed under the help of liposome. Positive PT67 cells were picked out with G418, and proliferated for harvesting virus. Based on the titre of virus, after BMSCs were infected by virus containing pLEGFP-N1, GFP positive BMSCs were collected and proliferated for seeding cells. TEB was fabricated by GFP positive BMSCs and decalcified bone matrix (DBM) and observed by CLSM-3DR for the evaluation of the distribution and proliferation of seeding cells. After TEB was transplanted in the defect of goat femur, CLSM was used for observing the survival and distribution of GFP positive cells in the grafts., Results: The isolated cells were fibroblast-like amorphous, with the positive expression of CD29 and CD44, and negative expression of CD60L and CD45. The digested production of pLEGFP-N1 was collected for ionophoresis, whose results showed the correct fragment length (6 900 bp). The virus of pLEGFP-N 1 was harvested by transfection of pLEGFP-N1 into PT67 cells and used for further infection to obtain GFP positive BMSCs. The proliferated GFP positive BMSCs and DBM were used for fabrication of TEB. The distribution, proliferation, and migration of BMSCs in TEB were observed by CLSM-3DR. GFP positive cells also were observed in images of TEB graft in goat femur 28 days after transplantation., Conclusion: The BMSCs labeled by GFP in three-dimensional scaffold in vivo were monitored well by CLSM-3DR. It suggests a wide use potency in monitoring of three-dimensional cultured TEB.

Although bone marrow-derived mesenchymal stromal cells (BMSCs) are a main cell source for tissue-engineered bone (TEB), the clinical use of BMSCs is restricted due to the invasive bone marrow aspiration procedure and the decline in available number of mesenchymal stromal cells (MSCs) and differentiation potential with increasing age. Umbilical cord-derived MSCs (UCMSCs) are likely to be a promising alternative cell source for TEB due to their higher availability and potential to proliferate and differentiate. To assess this possibility, we studied bone morphogenetic protein 2 (BMP2)-induced osteogenic differentiation and activation of signaling pathways in UCMSCs and BMSCs. UCMSCs showed a phenotype and differentiation potential similar to that of BMSCs. After 14 days of BMP2 treatment, the overall expression of several osteogenic-specific phenotypes (type I collagen, osteopontin, and osteocalcin) was similar for UCMSCs and BMSCs. The signaling pathway by which BMP2 induced differentiation of both cell types involved the membrane receptor-initiated signals including SMADs, P38, and extracellular regulated kinase. The similar characteristics of BMP2-induced osteogenic differentiation of UCMSCs and BMSCs in vitro would support the use of UCMSCs in TEB.

Background/Aims: Bone resorption mediated by osteoclasts plays an important role in bone healing. Endothelial progenitor cells (EPCs) promote bone repair by stimulating neovascularization and osteogenesis. However, the role of EPCs in osteoclast formation and function is not well defined. The aim of this study was to elucidate mechanisms of EPCs in osteoclast formation and function. Methods: In this study, we examined the effects of EPCs on the proliferation, migration and osteoclastic differentiation of primary mouse bone marrow-derived macrophages (BMMs) in a co-culture system in vitro. We also evaluated the effects of EPC co-transplantation on the homing and osteoclastic differentiation of transplanted BMMs in a mouse bone fracture model in vivo. The technology of immunofluorescence, immunohistochemical, western blot, Rt-PCR, cell co-culture and Transwell were used in this study. Results: EPCs secreted TGF-β1 in the EPC-BMM co-culture medium and increased Talin-1 expression in the co-cultured BMMs. Treatment with a TGF-β1 neutralizing antibody or Talin-1 silencing in BMMs completely inhibited BMM osteoclastic differentiation in the co-culture system. These results indicated that the osteoclastogenic effects of EPCs were mediated by TGF-β1-mediated Talin-1 expression in BMMs. In the femur fracture model, BMMs co-transplanted with EPCs exhibited enhanced engraftment into the fracture site and osteoclastic differentiation compared with those transplanted alone. Mice treated with EPC-BMM co-transplantation exhibited increased neovascularization at the fracture site and accelerated fracture healing compared with those treated with BMMs alone. Conclusion: Taken together, the results suggest that EPCs can promote bone repair by enhancing recruitment and differentiation of osteoclast precursors. [ABSTRACT FROM AUTHOR]

Purpose: This study focuses on nanoscale self-assembly peptides (SAP) modified demineralized bone matrix (DBM) which provided a more effective osteogenesis and regeneration for critically-sized femur defects in goats using the selective cell retention (SCR) strategy. Methods: RADA16-I peptide was used to modify DBM and formed a composite scaffold (SAP/DBM). The morphological change and dynamic expression of osteogenic genes of mesenchymal stem cells (MSCs) derived from marrow in SAP/DBM was observed. The cells and factors in bone marrow were enriched into SAP/DBM by technology of selective cells retension (SCR). The construct was transplanted into 20-mm femur defects in goats and their osteogenesis was evaluated. Results: The SAP/DBM scaffold formed a three-dimensional interweaving nanofiber in pores of DBM. MSCs exhibited better morphology in SAP/DBM than that in only DBM, and the levels of expression of ALP ,OCN and Runx2 gene in SAP/DBM samples was significantly higher than that of DBM at 14 days in vitro ( P < 0.05). Compared with marrow-enriched DBM, the volume of newly formed bone from marrow-enriched SAP/DBM is higher in goats ( P < 0.05). Conclusion: Our study may not only have a significant impact on the construction method of tissue engineering but also provide a viable, simple and effective method for clinical bone construction. [ABSTRACT FROM AUTHOR]