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      Use of a biological reactor and platelet-rich plasma for the construction of tissue-engineered bone to repair articular cartilage defects

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          Abstract

          Articular cartilage defects are a major clinical burden worldwide. Current methods to repair bone defects include bone autografts, allografts and external fixation. In recent years, the repair of bone defects by tissue engineering has emerged as a promising approach. The present study aimed to assess a novel method using a biological reactor with platelet-rich plasma to construct tissue-engineered bone. Beagle bone marrow mesenchymal stem cells (BMSCs) were isolated and differentiated into osteoblasts and chondroblasts using platelet-rich plasma and tricalcium phosphate scaffolds cultured in a bioreactor for 3 weeks. The cell scaffold composites were examined by scanning electron microscopy (SEM) and implanted into beagles with articular cartilage defects. The expression of osteogenic markers, alkaline phosphatase and bone γ-carboxyglutamate protein (BGLAP) were assessed using polymerase chain reaction after 3 months. Articular cartilage specimens were observed histologically. Adhesion and distribution of BMSCs on the β-tricalcium phosphate (β-TCP) scaffold were confirmed by SEM. Histological examination revealed that in vivo bone defects were largely repaired 12 weeks following implantation. The expression levels of alkaline phosphatase (ALP) and BGLAP in the experimental groups were significantly elevated compared with the negative controls. BMSCs may be optimum seed cells for tissue engineering in bone repair. Platelet-rich plasma (PRP) provides a rich source of cytokines to promote BMSC function. The β-TCP scaffold is advantageous for tissue engineering due to its biocompatibility and 3D structure that promotes cell adhesion, growth and differentiation. The tissue-engineered bone was constructed in a bioreactor using BMSCs, β-TCP scaffolds and PRP and displayed appropriate morphology and biological function. The present study provides an efficient method for the generation of tissue-engineered bone for cartilage repair, compared with previously used methods.

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          Platelet-rich plasma: evidence to support its use.

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            Platelet-rich plasma: Growth factor enhancement for bone grafts.

            Platelet-rich plasma is an autologous source of platelet-derived growth factor and transforming growth factor beta that is obtained by sequestering and concentrating platelets by gradient density centrifugation. This technique produced a concentration of human platelets of 338% and identified platelet-derived growth factor and transforming growth factor beta within them. Monoclonal antibody assessment of cancellous cellular marrow grafts demonstrated cells that were capable of responding to the growth factors by bearing cell membrane receptors. The additional amounts of these growth factors obtained by adding platelet-rich plasma to grafts evidenced a radiographic maturation rate 1.62 to 2.16 times that of grafts without platelet-rich plasma. As assessed by histomorphometry, there was also a greater bone density in grafts in which platelet-rich plasma was added (74.0% +/- 11%) than in grafts in which platelet-rich plasma was not added (55.1% +/- 8%; p = 0.005).
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              The in vitro effect of different PRP concentrations on osteoblasts and fibroblasts.

              The aim of this study was to assess the biological rationale for the use of platelet-rich plasma (PRP) by evaluating the effect of different concentrations of PRP on osteoblasts (OB) and fibroblasts (FB) function in vitro. PRP was obtained from volunteer donors using standard protocols. Primary human cultures of oral FBs and OBs were exposed to both activated and non-activated plasma as well as various concentrations of PRP (2.5 x, 3.5 x and max (4.2-5.5 x)). Cell proliferation was evaluated after 24 and 72 h using an MTT proliferation assay. Production of osteocalcin (OCN), osteoprotegerin (OPG) and transforming growth factor beta1 (TGF-beta1) was evaluated in OB after 24 and 72 h. Statistical analysis was performed using one-way ANOVA. PRP-stimulated cell proliferation in both OBs and FBs. The effect of different PRP concentrations on cell proliferation was most notable at 72 h. The maximum effect was achieved with a concentration of 2.5 x, with higher concentrations resulting in a reduction of cell proliferation. Upregulation of OCN levels and downregulation of OPG levels were noted with increasing PRP concentrations at both 24 and 72 h. TGF-beta1 levels were stimulated by increasing concentrations of PRP, with the increased levels being maintained at 72 h. PRP preparations exert a dose-specific effect on oral FBs and OBs. Optimal results were observed at a platelet concentration of 2.5 x, which was approximately half of the maximal concentrate that could be obtained. Increased concentrations resulted in a reduction in proliferation and a suboptimal effect on OB function. Hence, different PRP concentrations may have an impact on the results that can be obtained in vivo.
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                Author and article information

                Journal
                Exp Ther Med
                Exp Ther Med
                ETM
                Experimental and Therapeutic Medicine
                D.A. Spandidos
                1792-0981
                1792-1015
                August 2016
                23 May 2016
                23 May 2016
                : 12
                : 2
                : 711-719
                Affiliations
                [1 ]Department of Orthopedics, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, P.R. China
                [2 ]Department of Orthopedics, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, Shandong 250021, P.R. China
                Author notes
                Correspondence to: Professor Hao Liu, Department of Orthopedics, West China Hospital, Sichuan University, 37 Guoxue Road, Chengdu, Sichuan 610041, P.R. China, E-mail: liuhao6304@ 123456163.com
                Article
                ETM-0-0-3380
                10.3892/etm.2016.3380
                4950899
                27446265
                78c2daa8-e7b9-4886-922e-e20425807696
                Copyright: © Li et al.

                This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

                History
                : 25 May 2015
                : 14 April 2016
                Categories
                Articles

                Medicine
                bone repair,bone tissue engineering,bone regeneration,bioreactor,platelet-rich plasma
                Medicine
                bone repair, bone tissue engineering, bone regeneration, bioreactor, platelet-rich plasma

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