In Vitro Evaluation of the Allogeneic Bone Matrix Effect on the Adipose Mesenchymal Stromal Cells Characteristics in Combined Tissue Engineering

The aim of the study was to evaluate in vitro the effect of native and deproteinized compact and spongy allogenic bone matrices on the characteristics of adipose mesenchymal stromal cells (ASC) in combined tissue engineering.Material and Methods. 24 samples of native and deproteinized compact and spongy bone were examined, which were exposed to mechanical treatment, modeling, followed by sterilization of the samples by ionizing radiation and bacteriological control of sterilization. Some of the samples underwent deproteinization. The characterized cultures of human ASC were used as test cultures to assess the interaction with the bone samples. The Cytation-5 fluorescent imager and Hoechst 3334 fluorochromes (BD Pharmingen™) and calcein (Calcein AM, BD Pharmingen™) were used to characterize the degree of adhesion, migration, and viability of ASC on bone matrix samples. Matrix cytotoxicity was evaluated by MTT assay on days 1 and 7 of extraction.Results. The bone matrix samples are characterized by the absence of cytotoxicity (rank 1). ASC demonstrated good adhesion and migration on any surface of the bone matrix and preservation of cell viability during 7 days of observation. Nuclei sizes of the cells adhered to the deproteinized bone matrix of the spongy structure increased by 25–30% compared to other samples. The cells on deproteinized bone matrix had greater size (the size of the cells from nuclei 8.8 to 11.5 μm, the average size of cells nuclei from an 86.3 μm to 129,0 μm, the average perimeter of the cells nuclei from 30.7 μm to 40.7 μm) than in the native bone matrix samples.Conclusion. The results of the study of various allogeneic bone matrices demonstrate that deep purification of the bone matrix determines the absence of cytotoxicity and the most favorable conditions for the adhesion, migration, proliferation and viability of ASC. Also makes it possible to use tissue engineering based on bone matrices of different structures. Deproteinized spongy bone matrices are best suited for this purpose.

Repair of segmental bone defect using tissue engineered heterogeneous deproteinized bone doped with lithium

Lithium have been shown to play an important role in improving the osteogenic properties of biomaterials. This study aims to explore the osteogenic improvement effect of tissue engineered heterogeneous deproteinized bone (HDPB) doped with lithium, and evaluate their effectiveness in the healing of bone defects. Bone marrow mesenchymal stem cells (BMSCs) were co-cultured with different concentration of lithium chloride. Cell proliferation in each group was analyzed by 3-(4, 5-dimetyl-2-thiazoly-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) assay. BMSCs were then co-cultured in osteogenic induction medium with different concentration of lithium chloride, and the expression of related mRNA was detected. The role of lithium in promoting BMSCs osteogenic differentiation and inhibiting BMSCs lipogenic differentiation was also investigated. Biomechanical properties of the tibia were evaluated at 8 weeks after operation. The tibial specimens of each group were collected at 4 and 8 weeks after surgery for histological examination and histological analysis. Micro-computed tomography (CT) scanning and 3D reconstruction were performed at 8 weeks. The results demonstrate that lithium can induce the osteogenic differentiation inhibit of adipogenic differentiation of BMSCs by regulating the Wnt signaling pathway. The histological evaluation further certified that average bone formation area in the group of tissue engineered HDPB doped with lithium was also significantly better than that of HDPB alone group. Based on the above evaluation, tissue engineered HDPB doped with lithium can effectively promote the regeneration of segmental bone defect, which can be used as a tissue engineering scaffold for clinical trials.

The assessment of xenogeneic bone immunotoxicity and risk management study

Background Xenogeneic bone has been widely used in a variety of clinical bone-related disease to promote bone healing and restore bone defects. However, the adverse effects of immune system limit its application in the clinic. The aim of this study was to evaluate xenogeneic bone safety of immunotoxicity and explore the methods for immune risk supervision. Results Xenogeneic bone, which is freeze-dried bovine cancellous bone, was implanted into the muscle of mice. On day 7, 14 and 28, the effects of xenogeneic bone were examined on humoral immunity and cellular immunity, including the levels of IgG, IgM, C3, inflammatory factors (TNF-α, IL-6), alkaline phosphatase (ALP) and the lymphocyte phenotype. The data showed that xenogeneic bone implantation had no potential to induce immune responses not only in humoral immunity but also in cellular immunity. To reveal the risk of immunogenicity, the residual DNA and the clearance of α-gal epitope were analyzed in 2 different bones (bone 1 is deproteinized bone, bone 2 is acellular and defatted bone). It was suggested that DNA of xenogeneic bone can be limited to < 50 ng per mg dry weight for the repair or regeneration with the acceptable immune risk. And α-gal clearance of xenogeneic bone could be an effective risk factor for improving xenograft quality management. Conclusions Through the detection of xenogeneic bone immunotoxicity, our findings indicated that the supervisions of risk factors could contribute to reduce the immune risk. And the risk factors under the acceptable limitation could decrease or replace animal experiment. However, it still needs to be studied on the limitation of α-gal epitope to predict rejection of xenogeneic bone more accurately.