Bone marrow aspirate concentrate (BMAC) including high densities of stem cells and progenitor cells may possess a stronger bone regenerative capability compared with Platelet-rich plasma (PRP), which contains enriched growth factors. The objective of this study was to evaluate the effects of human BMAC and PRP in combination with β-tricalcium phosphate (β-TCP) on promoting initial bone augmentation in an immunodeficient mouse model.
BMAC and PRP were concentrated with an automated blood separator from the bone marrow and peripheral blood aspirates. β-TCP particles were employed as a scaffold to carry cells. After cell counting and FACS characterization, three groups of nude mice (BMAC+TCP, PRP+TCP, and a TCP control) were implanted with graft materials for onlay placement on the cranium. Samples were harvested after 4 weeks, and serial sections were prepared. We observed the new bone on light microscopy and performed histomorphometric analysis. After centrifugation, the concentrations of nucleated cells and platelets in BMAC were increased by factors of 2.8±0.8 and 5.3±2.4, respectively, whereas leucocytes and platelets in PRP were increased by factors of 4.1±1.8 and 4.4±1.9, respectively. The concentrations of CD34-, CD271-, CD90-, CD105-, and CD146-positive cells were markedly increased in both BMAC and PRP. The percentage of new bone in the BMAC group (7.6±3.9%) and the PRP group (7.2±3.8%) were significantly higher than that of TCP group (2.7±1.4%). Significantly more bone cells in the new bone occurred in sites transplanted with BMAC (552±257) and PRP (491±211) compared to TCP alone (187±94). But the difference between the treatment groups was not significant.
Citation: Zhong W, Sumita Y, Ohba S, Kawasaki T, Nagai K, Ma G, et al. (2012) In Vivo Comparison of the Bone Regeneration Capability of Human Bone Marrow Concentrates vs. Platelet-Rich Plasma. PLoS ONE 7(7): e40833. https://doi.org/10.1371/journal.pone.0040833
Editor: Nuno M. Neves, University of Minho, Portugal
Received: November 9, 2011; Accepted: June 15, 2012; Published: July 12, 2012
Copyright: © 2012 Zhong et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study has been partially supported by the Grant-in-Aid for Scientific Research (22390387) from Japan Society for the Promotion of Science. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
The regeneration and reconstruction of missing bone in patients with persistent bone defects may be difficult to achieve without interventions such as bone grafting. Different techniques utilizing autologous bone and allografts, xenografts, and various artificial bone substitutes have been developed. However, these techniques have drawbacks and have shown limited success . The need for a more effective regenerative approach led to the development of tissue engineering techniques that usually involve one or more of the following three key elements: scaffold or supporting matrices; growth factors or signaling molecules; and cells . Because only a small amount of tissue from the patient is required, bone reconstruction with this technique is less invasive and safer than conventional methods.
Platelet-rich plasma (PRP) enhances osteogenesis and accelerates healing of an existing wound because growth factors are released from platelets after the coagulation process is locally triggered in the wound site –. The growth factors produced by human platelets include platelet-derived growth factor, insulin-like growth factor, transforming growth factor β, basic fibroblast growth factor, epidermal growth factor, and vascular endothelial growth factor , . Hence, the use of PRP may not only improve and facilitate the manipulation of particulate grafts but also increase vascular ingrowth and mitogenic effects on bone-forming cells , .
The stem cells and progenitor cells derived from bone marrow are the most useful sources of autologous cells for bone tissue regeneration –. Recently, bone marrow aspirate concentrate (BMAC) was suggested to contain an enriched population of mononuclear cells (MNCs) and cytokines and has attracted the attention of clinicians . Utilization of BMAC may bypass the time-consuming and technically difficult process of cell expansion and differentiation, enabling both harvesting and transplanting of BMAC during the same surgical procedure . Furthermore, the platelets in BMAC may provide conditions permitting more rapid and effective bone regeneration by mesenchymal stem cells (MSCs).
Well-controlled comparative studies regarding the bone regenerative capability of these two concentrates isolated from peripheral blood and bone marrow remain scarce, and the results are controversial. A clinical study demonstrated that peripheral blood PRP possesses better potential for alveolar bone augmentation compared with bone marrow-derived cells . Conversely, a recent experimental study claimed that PRP shows no beneficial effects on bone formation and that bone marrow MNCs display significant positive effects on bone regeneration compared to PRP .
To explore a feasible approach for facilitating the clinical application of bone tissue engineering techniques, the bone regenerative capabilities of human BMAC and peripheral blood PRP were evaluated with an immunodeficient mouse model using β-tricalcium phosphate (β-TCP) as a scaffold. The bone regeneration effects were evaluated histologically after 4 weeks of healing.
The concentration of bone marrow nucleated cells increased by a factor of 2.8±0.8 from 19.8±8.2×106/ml to 58.8±33.6×106/ml. White blood cells in peripheral blood increased by a factor of 4.1±1.8 after centrifugation from 4.9±1.5×106/ml to 18.4±5.2×106/ml. Platelets were enriched by a factor of 5.3±2.4 in bone marrow from 12.9±5.2×107/ml to 67.2±37.6×107/ml, and by 4.4±1.9 times in peripheral blood from 18.4±3.8×107/ml to 76.7±25.0×107/ml (Table 1).
No significant differences were found between the rates of cell concentration increase in bone marrow and peripheral blood (p = 0.25). Although the cell concentrations were variable among donors before and after centrifugation, similar tendencies were exhibited. No significant correlations were found between cell recovery rate and age or gender.
FACS analysis showed that the cell populations from bone marrow and peripheral blood were both positive for well-known stem cell markers including CD34, CD90, CD105, CD271, and CD146 (Figure 1). The proportion of FITC-positive cells in the isotype control was less than 0.3%. Over 92% of bone marrow cells and isolated cell products were positive for CD45. After centrifugation, the percentage of the different stem cells varied in relation to donor and cell categories, possibly reflecting the heterogeneity of the original population (Table S1).
The data of CD34- and CD105-positive cells in bone marrow and the associated isolated fractions came from donor 1. No apparent changes were present in the proportion of CD34- (6.7% to 6.8%) and CD105- (19.3% to 19.3%) positive cells between bone marrow and the BMAC fractions post-isolation. The proportions of FITC-positive cells in the isotype controls were 0.1% and 0.2%, respectively.
Generally, the proportions of CD marker positive cell changed slightly after centrifugation according to the averaged FACS values (Table 2). No significant statistical differences were found between CD marker positive cell proportion before and after centrifugation.
Four weeks after implantation, a small amount of new bone was found in the immediate proximity of the host bone in the TCP control group (Figure 2A). However, the augmented area was mostly filled with β-TCP particles, which were embedded in loose connective tissue, without bone formation. In the PRP and BMAC groups, considerably more new bone was present in areas close to the host bone, and the new bone surrounded the β-TCP particles and connected with the host bone (Figure 2B, 2C). Newly formed bone was also observed in the macropores of the β-TCP particles. Blood vessels could be observed throughout the specimens,