Several TRAP-positive, multinucleated cells were observed along the surface of the MBCP and MBCP plus particles (Fig. 6A), while there were almost no TRAP-positive cells on the Bio-Oss particles. In particular, small particles in the MBCP plus group were densely surrounded by multinucleated cells, and some exhibited typical resorption pits. The greatest number of cells was observed in the MBCP plus group, and the lowest was found in the Bio-Oss group. The differences between all of the carrier groups were statistically significant (P<0.05).
IV. Discussion
The results of this study show that the use of BCP ceramics as a cell carrier for hABMSCs resulted in a significantly greater degree of bone formation in the ectopic transplantation model with than Bio-Oss. In addition, Bio-Oss was associated with significantly lower induction of osteoclasts along the surface compared to MBCP and MBCP plus. A greater osteogenic differentiation was exhibited by hABMSCs loaded onto MBCP and MBCP plus carriers, as corroborated by immunohistochemical analyses.
It is well established that both Bio-Oss and HA/TCP carriers are recommended for dental application as an alternative to autogenous bone due to their biocompatibility and higher osteoconductive potential.(Araujo et al 2008; Hench & Polak 2002;
Mordenfeld et al 2010; Schopper et al 2005; Schwartz et al 1999) However, indications of those previous studies were limited to bone defects in which adjacent bone tissues were able to provide the osteogenic source. The present results--limited osteogenecity with Bio-Oss--were not in accordance with these studies. This could have arisen from the present experimental model of ectopic transplantation, which can provide only limited sources of healing from the surrounding connective tissue.
This experimental model is mainly used to evaluate the multipotentiality of cells or osteoinductivity of growth factors by ectopic formation of bone, cartilage, cementum, or ligament from stem cells or growth factor.
The neogenesis in an ectopic site is dependent on the type of related cells, signals, and the characteristics of the carrying scaffold. Our previous study demonstrated different de novo tissue formation by periodontal ligament stem cells with/without bone morphogenetic protein-2 (cementum/periodontal ligament or bone/adipose tissue by the presence of BMP-2).(Lee et al 2014; Song et al 2011) In addition, another of our previous studies found specific collagen formation by stem-cell-carrying hyaluronic acid despite the use of the same periodontal ligament stem cells as in the aforementioned study.(Park et al 2015) The present study also demonstrated different healing patterns dependent on the type of carrying scaffolds: de novo bone formation by hABMSC/BCP but minimal formation by hABMSC/Bio-Oss.
The osteogenic potential of BCP-carrying multipotent stem cells using the ectopic transplantation model has already been noted in many previous in vitro studies, whereas in in vivo studies, they were found to facilitate bone ingrowth.(Saldana et al 2009; Schwartz et al 1999; Sun et al 1997) Arinzhe et al. reported that altering the composition of HA/TCP may influence the amount of bone formation, and demonstrated that when loaded with human MSCs, do novo bone formation in the mouse ectopic model was increased dependently by the proportion of TCP in BCP.(Arinzeh et al 2005) These findings were in line with the present immunohistochemistry result that the MBCP plus group showed significantly more positively stained cells for ALP, RUNX-2, OCN, and OPN, compared to the MBCP as well as the Bio-Oss group. (The MBCP group also showed a greater number of positive cells for the aforementioned osteogenic markers compared to the Bio-Oss
group.) Higher rates of degradation in these carriers would presumably favor the osteogenic potential since the superficial degradation of TCP increases the concentrations of calcium ions along the surfaces, and this calcium-rich microenvironment promotes the differentiation of MSCs, expression of calcium-binding proteins, and calcium incorporation into the extracellular matrix.(Duncan et al 1998; Dvorak et al 2004; Lee et al 2014) In addition, the TRAP staining in the present study also revealed minimal TRAP-positive osteoclast formation with the Bio-Oss carrier, while actively resorbing particles surrounded by numerous osteoclasts were observed with MBCP and MBCP plus (and especially so in the latter). These findings confirm that the microenvironment of scaffolds significantly affected the behavior of hABMSCs and osteoclasts, which are in a coupled relationship.
Bio-Oss is the most widely used and studied biomaterial for dental bone tissue engineering in both clinical and research fields, in which successful results are mainly based on osteoconductivity as well as specific surface characteristics; the dimensions of the grafted biomaterial particles were maintained even at eleven years after surgery,(Mordenfeld et al 2010) and several previous studies found that the surface microstructures of Bio-Oss affected deposition of osteogenic proteins and direct bone formation onto the surfaces.(Araujo et al 2008; Hofman et al 1999; Mladenovic et al 2013; Orsini et al 2005) In this study, hABMSCs were able to attach at very high rates onto the surfaces of all experimental biomaterials (MBCP, 97.89±0.65; MBCP plus, 97.78±1.50; and Bio-Oss, 93.26±2.35), although there were statistically significant
differences between MBCP/MBCP plus and Bio-Oss). However, the present in vivo results showed that the Bio-Oss particles failed to promote hABMSC-induced bone formation as much as MBCP/MBCP plus scaffolds in histology, possibly as a result of different differentiation patterns of the cells in response to the surface characteristics of scaffolds, like isolation of calcium ions from HA/TCP particles.
These findings confirm that the microenvironment (including concentrations of calcium ions) of scaffolds significantly affected the behavior of hABMSCs and osteoclasts, which are in a coupled relationship.
V. Conclusion
As hABMSCs carriers, MBCP (20% HA/80% TCP) and MBCP plus (60%
HA/40% TCP) exhibited equally and significant osteoinductive potential in the ectopic transplantation model, as evidenced by histomorphometric and immunohistomorphometric analysis, with significantly increased osteoclast formation.
While Bio-Oss failed to induce new bone formation when loaded with hABMSCs, it was associated with minimal osteoclast formation along the surface. It therefore appears that both types of scaffolds, BCP and Bio-Oss, showed high stem cell-carrying potential, but the in vivo healing patterns of their complexes with hABMSC could be affected by the microenvironment on the surfaces of the scaffolds, like calcium ions from degraded TCP.
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Figure Legends
Figure 1.
Pore sizes of the carriers for human alveolar-bone-derived mesenchymal stem cells (hABMSCs) as measured using micro-computed tomography (CT). MBCP, macroporous biphasic calcium phosphate, MBCP plus, macroporous biphasic calcium phosphate plus. (A) Reconstructed micro CT images showing different pore composition of three different scaffolds. (B) The distribution of pore sizes was measured and compared. (C) Mean pore diameter was measured in the scaffolds.Figure 2.
Evaluation of the cell affinity of hABMSCs to scaffolds using scanning electron microscopy (SEM) and in vitro assay. (A) Cells were cultured overnight with each of the following scaffolds: MBCP, MBCP plus, and Bio-Oss, and SEM images were taken. (B) In vitro cell affinity assay revealed that MBCP and MBCP plus exhibited a significantly enhanced cell affinity to hABMSCs in comparison to Bio-Oss (*: P<0.05).Figure 3
. Ectopic transplantation assays were performed to evaluate the in vivo bone regeneration by hABMSCs loaded onto three different scaffolds. (A) Histologically, newly formed bone tissues were present along the periphery of the HA/TCP carriers, and osteocytes were noted inside the mineralized tissues (arrowheads). However, Bio-Oss was associated with minimal bone formation and no osteoblast lining on the particle. Hematoxylin and eosin (H&E) stain (Upper row: Scale bar= 200 μm, Lowerrow: Scale bar= 50 μm). MBCP and MBCP+ were associated with (B) significantly enhanced bone regeneration and (C) the formation of numerous osteocytes within the mineralized tissues (*: P<0.05). However, ABMSCs loaded onto Bio-Oss failed to produce any significant bone formation.
Figure 4.
Immunohistochemical staining of the ectopic transplantation model. (A) hABMSCs loaded onto MBCP particles were associated with newly formed bone tissue and cells stained positively for antibodies (arrowheads) raised against human mitochondria (hMito), proliferating cell nuclear antigen (PCNA), and collagen type I (Col I). hABMSCs loaded onto MBCP plus particles also exhibited bone formation with positively stained cells, comparable with those observed for MBCP. hABMSCs loaded onto Bio-Oss failed to show the presence of positively stained cells (Scale bar= 50 μm).
Figure 5.
Immunohistomorphometric analysis of hABMSCs loaded onto various carriers using the ectopic transplantation model. (A) Cells were stained against the following markers of osteogenesis: ALP, RUNX-2, OCN, and OPN. Positively stained cells were observed in the hABMSCs-loaded MBCP and hABMSCs-loaded MBCP plus groups (arrowheads). Positively stained cells were not observed in Bio-Oss group. (B) The number of positively stained cells were counted and illustrated.MBCP plus group showed statistically significant increase in positively-stained cells (*: P<0.05).
Figure 6.
(A) Tartrate-resistant acid phosphatase (TRAP) staining revealed the presence of multinucleated osteoclasts along the surfaces of the MBCP and MBCP plus particles (arrowheads). (B) There were significantly fewer osteoclasts in the Bio-Oss group when compared than with other carriers (*P<0.05).Figures
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