Foot fat pad: Characterization by mesenchymal stromal cells in rats

Foot fat pad (FFP) is a highly functionalized fat depot of great significance for weight bearing in the foot. Mesenchymal stromal cells (MSCs) in subcutaneous adipose tissues are widely studied for regenerative potentials. MSCs in FFP, which may contribute to the physiological and pathological conditions of the foot, have not been characterized. In this study, MSCs were isolated from FFP (designated as MSCs-ffp) and subcutaneous adipose tissue (designated as MSCs-sub) from rats. The cell surface markers, proliferation, and efficiency of colony formation were compared between MSCs-ffp and MSCs-sub. In addition, MSCs-ffp were induced for osteogenic, chondrogenic, and adipogenic differentiation. The tri-lineage differentiation potentials were compared between MSCs-ffp and MSCs-sub by the expression of Runx2, Sox9, and proliferator-activated receptor gamma (PPAR-γ), respectively, using quantitative polymerized chain reaction. The expression of elastin and associated genes by MSCs-ffp were also evaluated. MSCs-ffp, like MSCs-sub, expressed CD44, CD73, and CD90. MSCs-ffp and MSCs-sub proliferated at similar rates but MSCs-ffp formed more colonies than MSCs-sub. MSCs-ffp were capable of differentiating into osteogenic, chondrogenic, and adipogenic lineages. Under the conditions of osteogenic and adipogenic differentiation, MSCs-sub expressed more Runx2 and PPAR-γ, respectively, than MSCs-ffp. The undifferentiated MSCs-ffp upregulated the expression of fibulin-5. In conclusion, MSCs-ffp shared common biology with MSCs-sub but were more efficient in colony formation, less adipogenic and osteogenic, and participated in elastogenesis. The unique features of MSCs-ffp may relate to their roles in the physiological functions of FFP.     

Breast and abdominal adipose multipotent mesenchymal stromal cells and stage-specific embryonic antigen 4 expression

We assessed human mesenchymal stem cells (MSCs) harvested from breast and abdominal adipose tissues enriched in embryonic stage-specific antigen (SSEA-4) expression for osteogenic and adipogenic differentiation in comparison to a mixed cell population. Human adipose was obtained from abdominal and breast tissues of females undergoing gastric bypass and breast reduction, respectively. SSEA-4-expressing cells were enriched from the mixed cell population by magnetic cell sorting and expanded in culture. The results showed that freshly isolated cells from breast and abdominal tissues based on adipose from 3 patients comprised 12 and 10% SSEA-4+ cells, respectively. At passage 0, 48% of the cells from breast adipose tissue were positive for SSEA-4 while 12% of the cells from abdominal adipose tissue were positive for this antigen. The level of SSEA-4-expressing cells remained relatively constant with passaging; SSEA-4-expressing cells from breast tissue comprised 45% of the total while 27% of the cells from abdominal adipose tissue expressed SSEA-4 at passage 5. Cells sorted for SSEA-4 expression exhibited a higher potential for differentiation toward osteogenic and adipogenc cell lineages in vitro when compared to a mixed population. Interestingly, SSEA-4 expression was lost upon differentiation, suggesting that the antigen marks a subpopulation of MSCs. Taken together, the data demonstrate that breast adipose tissue is highly enriched in a subpopulation of MSCs expressing SSEA-4 and suggest that SSEA-4 may be a marker of a subpopulation of MSCs with high potential for osteogenic and adipogenic differentiation.     

Adipose tissue-derived stem cells for cell therapy of airway allergic diseases in mouse

The purpose of this study was to compare murine mesenchymal stem cells (MSCs) isolated from bone marrow (BM) and adipose tissue (AT) for the selection of suitable MSCs in cell therapy of an airway allergic animal model. We compared MSCs of BALB/c mice derived from BM and AT with respect to proliferation potential, immunophenotype, and multilineage differentiation capacity. In proliferation potential, MSCs from AT (ASCs) showed higher fibroblastoid colony-forming units frequencies and colony-forming efficiency than MSCs from BM (BMSCs). The flow cytometry analysis showed that both ASCs and BMSCs expressed MSCs-related antigens (CD90 and CD105), whereas they did not express hematopoiesis-related antigens (CD45 and CD11b). There was no significant difference in adipogenic, osteogenic, and chondrogenic differentiation between the murine ASCs and BMSCs. In conclusion, the present study has shown that ASCs had higher CFU-F frequencies and colony-forming efficiency than BMSCs. ASCs and BMSCs presented a similar surface immunophenotype and multilineage differentiation capacity. Therefore, ASCs in BALB/c mice might be a more useful material for cell therapy of the airway allergic experiment due to the abundance, relatively easy harvesting and high proliferation potential.     

Human bronchial fibroblasts exhibit a mesenchymal stem cell phenotype and multilineage differentiating potentialities

Mesenchymal stem cells (MSCs) are multipotent cells able to differentiate along different pathways including chondrogenic, osteogenic and adipogenic lineages. MSCs with a fibroblast-like morphology have been identified in human fetal lung. However, their frequency and characterization in human adult lung have not been yet evaluated. Therefore, we analyzed the mesenchymal phenotype and differentiation ability of cultured human adult bronchial fibroblast-like cells (Br) in comparison with those of mesenchymal cell progenitors isolated from fetal lung (ICIG7) and adult bone marrow (BM212) tissues. Surface immunophenotyping by flow cytometry revealed a similar expression pattern of antigens characteristic of marrow-derived MSCs, including CD34 (-), CD45 (-), CD90/Thy-1 (+), CD73/SH3, SH4 (+), CD105/SH2 (+) and CD166/ALCAM (+) in Br, ICIG7 and BM212 cells. There was one exception, STRO-1 antigen, which was only weakly expressed in Br cells. Analysis of cytoskeleton and matrix composition by immunostaining showed that lung and marrow-derived cells homogeneously expressed vimentin and nestin proteins in intermediate filaments while they were all devoid of epithelial cytokeratins. Additionally, alpha-smooth muscle actin was also present in microfilaments of a low number of cells. All cell types predominantly produced collagen and fibronectin extracellular matrix as evidenced by staining with the monoclonal antibodies to collagen prolyl 4-hydroxylase and fibronectin isoforms containing the extradomain (ED)-A together with ED-B in ICIG7 cells. Br cells similarly to fetal lung and marrow fibroblasts were able to differentiate along the three adipogenic, osteogenic and chondrogenic mesenchymal pathways when cultured under appropriate inducible conditions. Altogether, these data indicate that MSCs are present in human adult lung. They may be actively involved in lung tissue repair under physiological and pathological circumstances.     

体外扩增过程中人骨髓间充质干细胞的增殖与分化规律

目的：系统考察体外扩增过程中人骨髓间充质干细胞（MSC）的增殖与分化规律，为MSC任组织修复以及细胞治疗中的应用提供参考、方法：以全骨髓贴壁法分离成人肋骨骨髓MSC，在相同条件下分别考察各代细胞形态、生长、表面标记、细胞周期、成骨、成软骨及成脂肪能力的变化情况。结果：随代次增加，MSC增殖能力、成骨、成脂肪能力均有所下降，而成软骨能力无明显降低；成骨、成软骨及成脂肪能乃均保持到细胞衰老。存扩增过程中，MSC始终保持较高的纯度，CD29、CD44、CD105的阳性率均在90％以上，CD14、CD34和CD45的阳性率均在4％以下、结论：在体外培养过程中MSC干细胞特性逐渐丢失，其中向骨、脂肪方向的分化潜能较软骨方向更易失去；而多向分化能力的保持较之自我更新能力更为持久。MSC在7代以前可作为基础研究及临床应用的良好对象。     

Comparative analysis of mesenchymal stem cells from bone marrow, cartilage, and adipose tissue

Mesenchymal stem cells (MSCs) isolated from bone marrow (BM), cartilage, and adipose tissue (AT) possess the capacity for self-renewal and the potential for multilineage differentiation, and are therefore perceived as attractive sources of stem cells for cell therapy. However, MSCs from these different sources have different characteristics. We compared MSCs of adult Sprague Dawley rats derived from these three sources in terms of their immunophenotypic characterization, proliferation capacity, differentiation ability, expression of angiogenic cytokines, and anti-apoptotic ability. According to growth curve, cell cycle, and telomerase activity analyses, MSCs derived from adipose tissue (AT-MSCs) possess the highest proliferation potential, followed by MSCs derived from BM and cartilage (BM-MSCs and C-MSCs). In terms of multilineage differentiation, MSCs from all three sources displayed osteogenic, adipogenic, and chondrogenic differentiation potential. The result of realtime RT-PCR indicated that these cells all expressed angiogenic cytokines, with some differences in expression level. Flow cytometry and MTT analysis showed that C-MSCs possess the highest resistance toward hydrogen peroxide -induced apoptosis, while AT-MSCs exhibited high tolerance to serum deprivation-induced apoptosis. Both AT and cartilage are attractive alternatives to BM as sources for isolating MSCs, but these differences must be considered when choosing a stem cell source for clinical application.     

A comprehensive analysis of the dual roles of BMPs in regulating adipogenic and osteogenic differentiation of mesenchymal progenitor cells

Pluripotent mesenchymal stem cells (MSCs) are bone marrow stromal progenitor cells that can differentiate into osteogenic, chondrogenic, adipogenic, and myogenic lineages. Several signaling pathways have been shown to regulate the lineage commitment and terminal differentiation of MSCs. Here, we conducted a comprehensive analysis of the 14 types of bone morphogenetic protein (BMPs) for their abilities to regulate multilineage specific differentiation of MSCs. We found that most BMPs exhibited distinct abilities to regulate the expression of Runx2, Sox9, MyoD, and PPARgamma2. Further analysis indicated that BMP-2, BMP-4, BMP-6, BMP-7, and BMP-9 effectively induced both adipogenic and osteogenic differentiation in vitro and in vivo. BMP-induced commitment to osteogenic or adipogenic lineage was shown to be mutually exclusive. Overexpression of Runx2 enhanced BMP-induced osteogenic differentiation, whereas knockdown of Runx2 expression diminished BMP-induced bone formation with a decrease in adipocyte accumulation in vivo. Interestingly, overexpression of PPARgamma2 not only promoted adipogenic differentiation, but also enhanced osteogenic differentiation upon BMP-2, BMP-6, and BMP-9 stimulation. Conversely, MSCs with PPARgamma2 knockdown or mouse embryonic fibroblasts derived from PPARgamma2(-/-) mice exhibited a marked decrease in adipogenic differentiation, coupled with reduced osteogenic differentiation and diminished mineralization upon BMP-9 stimulation, suggesting that PPARgamma2 may play a role in BMP-induced osteogenic and adipogenic differentiation. Thus, it is important to understand the molecular mechanism behind BMP-regulated lineage divergence during MSC differentiation, as this knowledge could help us to understand the pathogenesis of skeletal diseases and may lead to the development of strategies for regenerative medicine.     

间充质干细胞——现代组织工程的新资源

间充质干细胞 ( mesenchymal stem cells,MSC)存在于人类、鸟类、啮齿类等生物的骨髓中 ,它具有向骨、软骨、脂肪、肌肉及肌腱等组织分化的潜能。人们可利用它的这一特性建立多种细胞或组织的体外分化模型 ,从而为人类的细胞移植或组织移植提供可能的自体资源。本文就 MSC的生物学特性、体外分离方法、向各中胚层组织的分化条件及检测作一简要综述     

The effect of matrix stiffness on the differentiation of mesenchymal stem cells in response to TGF-β

Bone marrow mesenchymal stem cells (MSCs) are a valuable cell source for tissue engineering and regenerative medicine. Transforming growth factor β (TGF-β) can promote MSC differentiation into either smooth muscle cells (SMCs) or chondrogenic cells. Here we showed that the stiffness of cell adhesion substrates modulated these differential effects. MSCs on soft substrates had less spreading, fewer stress fibers and lower proliferation rate than MSCs on stiff substrates. MSCs on stiff substrates had higher expression of SMC markers α-actin and calponin-1; in contrast, MSCs on soft substrates had a higher expression of chondrogenic marker collagen-II and adipogenic marker lipoprotein lipase (LPL). TGF-β increased SMC marker expression on stiff substrates. However, TGF-β increased chondrogenic marker expression and suppressed adipogenic marker expression on soft substrates, while adipogenic medium and soft substrates induced adipogenic differentiation effectively. Rho GTPase was involved in the expression of all aforementioned lineage markers, but did not account for the differential effects of substrate stiffness. In addition, soft substrates did not significantly affect Rho activity, but inhibited Rho-induced stress fiber formation and α-actin assembly. Further analysis showed that MSCs on soft substrates had weaker cell adhesion, and that the suppression of cell adhesion strength mimicked the effects of soft substrates on the lineage marker expression. These results provide insights of how substrate stiffness differentially regulates stem cell differentiation, and have significant implications for the design of biomaterials with appropriate mechanical property for tissue regeneration.     

Multilineage cells from human adipose tissue: implications for cell-based therapies

Future cell-based therapies such as tissue engineering will benefit from a source of autologous pluripotent stem cells. For mesodermal tissue engineering, one such source of cells is the bone marrow stroma. The bone marrow compartment contains several cell populations, including mesenchymal stem cells (MSCs) that are capable of differentiating into adipogenic, osteogenic, chondrogenic, and myogenic cells. However, autologous bone marrow procurement has potential limitations. An alternate source of autologous adult stem cells that is obtainable in large quantities, under local anesthesia, with minimal discomfort would be advantageous. In this study, we determined if a population of stem cells could be isolated from human adipose tissue. Human adipose tissue, obtained by suction-assisted lipectomy (i.e., liposuction), was processed to obtain a fibroblast-like population of cells or a processed lipoaspirate (PLA). These PLA cells can be maintained in vitro for extended periods with stable population doubling and low levels of senescence. Immunofluorescence and flow cytometry show that the majority of PLA cells are of mesodermal or mesenchymal origin with low levels of contaminating pericytes, endothelial cells, and smooth muscle cells. Finally, PLA cells differentiate in vitro into adipogenic, chondrogenic, myogenic, and osteogenic cells in the presence of lineage-specific induction factors. In conclusion, the data support the hypothesis that a human lipoaspirate contains multipotent cells and may represent an alternative stem cell source to bone marrow-derived MSCs.     

Comparison of potentials of stem cells isolated from tendon and bone marrow for musculoskeletal tissue engineering

The use of tendon-derived stem cells (TDSCs) as a cell source for musculoskeletal tissue engineering has not been compared with that of bone marrow stromal cells (BMSC). This study compared the mesenchymal stem cell (MSC) and embryonic stem cells (ESC) markers, clonogenicity, proliferative capacity, and multilineage differentiation potential of rat TDSC and BMSC in vitro. The MSC and ESC marker profiles of paired TDSC and BMSC were compared using flow cytometry and quantitative real-time polymerase chain reaction (qRT-PCR), respectively. Their clonogenicity and proliferative capacity were compared using colony-forming and 5-bromo-2'-deoxyuridine assays, respectively. The expression of tenogenic, osteogenic, and chondrogenic markers at basal state were examined using qRT-PCR. Their osteogenic, chondrogenic, and adipogenic differentiation potentials were compared using standard assays. TDSC and BMSC showed similar expression of CD90 and CD73. TDSC expressed higher levels of Oct4 than BMSC. TDSC exhibited higher clonogenicity, proliferated faster, and expressed higher tenomodulin, scleraxis, collagen 1 α 1 (Col1A1), decorin, alkaline phosphatase, Col2A1, and biglycan messenger RNA levels than BMSC. There was higher calcium nodule formation and osteogenic marker expression in TDSC than BMSC upon osteogenic induction. More chondrocyte-like cells and higher glycosaminoglycan deposition and chondrogenic marker expression were observed in TDSC than BMSC upon chondrogenic induction. There were more oil droplets and expression of an adipogenic marker in TDSC than BMSC upon adipogenic induction. TDSC expressed higher Oct4 levels, which was reported to positively regulate mesendodermal lineage differentiation, showed higher clonogenicity and proliferative capacity, and had greater tenogenic, osteogenic, chondrogenic, and adipogenic markers and differentiation potential than BMSC. TDSC might be a better cell source than BMSC for musculoskeletal tissue regeneration.     

Ultrastructural and immunocytochemical analysis of multilineage differentiated human dental pulp- and umbilical cord-derived mesenchymal stem cells

Mesenchymal stem cells (MSCs) are one of the most promising stem cell types due to their availability and relatively simple requirements for in vitro expansion and genetic manipulation. Besides the well-characterized MSCs derived from bone marrow, there is growing evidence suggesting that dental pulp and the umbilical cord matrix both contain a substantial amount of cells having properties similar to those of MSCs. In order to assess the potential of dental pulp-derived MSCs (DPSC) and umbilical cord-derived MSCs (UCSC) in future clinical applications, it is essential to gain more insight into their differentiation capacity and to evaluate the tissues formed by these cells. In the present study, the morphological and ultrastructural characteristics of DPSC and UCSC induced towards osteogenic, adipogenic, and chondrogenic lineages were investigated. Cultured DPSC and UCSC showed a similar expression pattern of antigens characteristic of MSCs including CD105, CD29, CD44, CD146, and STRO-1. Under appropriate culture conditions, both DPSC and UCSC showed chondrogenic and osteogenic potential. Adipogenesis could be only partially induced in DPSC resulting in the de novo expression of fatty acid binding protein (FABP), whereas UCSC expressed FABP combined with a very high accumulation of lipid droplets in the cytoplasm. Our results demonstrate, at the biochemical and ultrastructural level, that DPSC display at least bilineage potential, whereas UCSC, which are developmentally more primitive cells, show trilineage potential. We emphasize that transmission electron microscopical analysis is useful to elucidate detailed structural information and provides indisputable evidence of differentiation. These findings highlight their potential therapeutic value for cell-based tissue engineering.     

Effect of Anatomical Origin and Cell Passage Number on the Stemness and Osteogenic Differentiation Potential of Canine Adipose-Derived Stem Cells

Mesenchymal stem cells have a great potential for application in cell based therapies, such as tissue engineering. Adipose derived stem cells have shown the capacity to differentiate into several lineages, and have been isolated in many animal species. Dog is a very relevant animal model to study several human diseases and simultaneously an important subject in veterinary medicine. Thus, in this study we assessed the potential of canine adipose tissue derived stem cells (cASCs) to differentiate into the osteogenic and chondrogenic lineages by performing specific histological stainings, and studied the cell passaging effect on the cASCs stemness and osteogenic potential. We also evaluated the effect of the anatomical origin of the adipose tissue, namely from abdominal subcutaneous layer and from greater omentum. The stemness and osteogenic differentiation was followed by real time RT-PCR analysis of typical markers of mesenchymal stem cells (MSCs) and osteoblasts. The results obtained revealed that cASCs exhibit a progressively decreased expression of the MSCs markers along passages and also a decreased osteogenic differentiation potential. In the author??s knowledge, this work presents the first data about the MSCs markers profile and osteogenic potential of cASCs along cellular expansion. Moreover, the obtained data showed that the anatomical origin of the adipose tissue has an evident effect in the differentiation potential of the ASCs. Due to the observed resemblances with the human ASCs, we conclude that canine ASCs can be used as a model cells in tissue engineering research envisioning human applications.     

Additive effects of sonic hedgehog and Nell-1 signaling in osteogenic versus adipogenic differentiation of human adipose-derived stromal cells

A theoretical inverse relationship exists between osteogenic (bone forming) and adipogenic (fat forming) mesenchymal stem cell (MSC) differentiation. This inverse relationship in theory partially underlies the clinical entity of osteoporosis, in which marrow MSCs have a preference for adipose differentiation that increases with age. Two pro-osteogenic cytokines have been recently studied that each also possesses antiadipogenic properties: Sonic Hedgehog (SHH) and NELL-1 proteins. In the present study, we assayed the potential additive effects of the biologically active N-terminus of SHH (SHH-N) and NELL-1 protein on osteogenic and adipogenic differentiation of human primary adipose-derived stromal cell (hASCs). We observed that both recombinant SHH-N and NELL-1 protein significantly enhanced osteogenic differentiation and reduced adipose differentiation across all markers examined (alkaline phosphatase, Alizarin red and Oil red O staining, and osteogenic gene expression). Moreover, SHH-N and NELL-1 directed signaling produced additive effects on the pro-osteogenic and antiadipogenic differentiation of hASCs. NELL-1 treatment increased Hedgehog signaling pathway expression; coapplication of the Smoothened antagonist Cyclopamine reversed the pro-osteogenic effect of NELL-1. In summary, Hedgehog and Nell-1 signaling exert additive effects on the pro-osteogenic and antiadipogenic differentiation of ASCs. These studies suggest that the combination cytokines SHH-N+NELL-1 may represent a viable future technique for inducing the osteogenic differentiation of MSCs.     

FGF-4 increases in vitro expansion rate of human adult bone marrow-derived mesenchymal stem cells

Human bone marrow-derived mesenchymal stem cells (MSCs) exhibit limited in vitro growth. Fibroblast growth factors (FGFs) elicit a variety of biological responses, such as cell proliferation, differentiation and migration. FGF-4 represents one of the FGFs with the highest cell mitogenic activity. We studied the effect of FGF-4 on MSCs growth and pluripotency. MSCs duplication time (Td) was significantly reduced with FGF-4 compared to controls (2.2 +/- 0.2 vs. 4.1 +/- 0.2 days, respectively; p = 0.03) while BMP-2 and SCF-1 did not exert a significant growth effect. MSC expression of surface markers, differentiation into adipogenic and osteogenic lineages, and baseline expression of cardiomyogenic genes were unaffected by FGF-4. In summary, exogenous FGF-4 increases the rate at which MSC proliferate and has no significant effect on MSC pluripotency.     

Identification of cord blood-derived mesenchymal stem/stromal cell populations with distinct growth kinetics, differentiation potentials, and gene expression profiles

Phenotypic heterogeneity has been observed among mesenchymal stem/stromal cell (MSC) populations, but specific genes associated with this variability have not been defined. To study this question, we analyzed two distinct isogenic MSC populations isolated from umbilical cord blood (UCB1 and UCB2). The use of isogenic populations eliminated differences contributed by genetic background. We characterized these UCB MSCs for cell morphology, growth kinetics, immunophenotype, and potential for differentiation. UCB1 displayed faster growth kinetics, higher population doublings, and increased adipogenic lineage differentiation compared to UCB2. However, osteogenic differentiation was stronger for the UCB2 population. To identify MSC-specific genes and developmental genes associated with observed phenotypic differences, we performed expression analysis using Affymetrix microarrays and compared them to bone marrow (BM) MSCs. We compared UCB1, UCB2, and BM and identified distinct gene expression patterns. Selected clusters were analyzed demonstrating that genes of multiple developmental pathways, such as transforming growth factor-beta (TGF-beta) and wnt genes, and markers of early embryonic stages and mesodermal differentiation displayed significant differences among the MSC populations. In undifferentiated UCB1 cells, multiple genes were significantly up-regulated (p < 0.0001): peroxisome proliferation activated receptor gamma (PPARG), which correlated with adipogenic differentiation capacities, hepatocyte growth factor (HGF), and stromal-derived factor 1 (SDF1/CXCL12), which could both potentially contribute to the higher growth kinetics observed in UCB1 cells. Overall, the results confirmed the presence of two distinct isogenic UCB-derived cell populations, identified gene profiles useful to distinguish MSC types with different lineage differentiation potentials, and helped clarify the heterogeneity observed in these cells.     

Maxillofacial-derived stem cells regenerate critical mandibular bone defect

Stem cell-based bone tissue regeneration in the maxillofacial complex is a clinical necessity. Genetic engineering of mesenchymal stem cells (MSCs) to follow specific differentiation pathways may enhance the ability of these cells to regenerate and increase their clinical relevance. MSCs isolated from maxillofacial bone marrow (BM) are good candidates for tissue regeneration at sites of damage to the maxillofacial complex. In this study, we hypothesized that MSCs isolated from the maxillofacial complex can be engineered to overexpress the bone morphogenetic protein-2 gene and induce bone tissue regeneration in vivo. To demonstrate that the cells isolated from the maxillofacial complex were indeed MSCs, we performed a flow cytometry analysis, which revealed a high expression of mesenchyme-related markers and an absence of non-mesenchyme-related markers. In vitro, the MSCs were able to differentiate into osteogenic, chondrogenic, and adipogenic lineages. Gene delivery of the osteogenic gene BMP2 via an adenoviral vector revealed high expression levels of BMP2 protein that induced osteogenic differentiation of these cells in vitro and induced bone formation in an ectopic site in vivo. In addition, implantation of genetically engineered maxillofacial BM-derived MSCs into a mandibular defect led to regeneration of tissue at the site of the defect; this was confirmed by performing micro-computed tomography analysis. Histological analysis of the mandibles revealed osteogenic differentiation of implanted cells as well as bone tissue regeneration. We conclude that maxillofacial BM-derived MSCs can be genetically engineered to induce bone tissue regeneration in the maxillofacial complex and that this finding may be clinically relevant.