Effects of FGF2 and FGF9 on osteogenic differentiation of bone marrow-derived progenitors

Bone repair is a major concern in reconstructive surgery. Transplants containing osteogenically committed mesenchymal stem cells (MSCs) provide an alternative source to the currently used autologous bone transplants which have limited supply and require additional surgery to the patient. A major drawback, however is the lack of a critical mass of cells needed for successful transplantation. The purpose of the present study was to test the effects of FGF2 and FGF9 on expansion and differentiation of MSCs in order to establish an optimal culture protocol resulting in sufficient committed osteogenic cells required for successful in vivo transplantation. Bone marrow-derived MSCs cultured in αMEM medium supplemented with osteogenic supplements for up to three passages (control medium), were additionally treated with FGF2 and FGF9 in various combinations. Cultures were evaluated for viability, calcium deposition and in vivo osteogenic capacity by testing subcutaneous transplants in nude mice. FGF2 had a positive effect on the proliferative capacity of cultured MSCs compared to FGF9 and control medium treated cultures. Cultures treated with FGF2 followed by FGF9 showed an increased amount of extracted Alizarin red indicating greater osteogenic differentiation. Moreover, the osteogenic capacity of cultured cells transplanted in immunodeficient mice revealed that cells that were subjected to treatment with FGF2 in the first two passages and subsequently to FGF9 in the last passage only, were more successful in forming new bone. It is concluded that the protocol using FGF2 prior to FGF9 is beneficial to cell expansion and commitment, resulting in higher in vivo bone formation for successful bone tissue engineering.     

Alterations of matrix- and cell-associated proteoglycans inhibit osteogenesis and growth response to fibroblast growth factor-2 in cultured rat mandibular condyle and calvaria

Matrix and cell surface proteoglycans (PGs) may play important roles in the control of cellular actions of heparan-binding growth factors such as fibroblast growth factor (FGF) during chondrogenesis and osteogenesis. In this study, we used 4-methylumbelliferyl-beta-d-xyloside, an inhibitor of PG synthesis, and sodium chlorate, a competitive inhibitor of glycoconjugate sulfation, to determine the functional consequences of alterations of PG metabolism on osteogenesis and on FGF actions in neonatal rat condyle and calvaria in vitro. Biochemical analysis showed that beta-d-xyloside (1 mM) or chlorate (15 mM) treatment for 1-8 days inhibited cellular PG synthesis by 60-80% in condyle and calvaria, as evaluated by [35S]sulfate incorporation. Histochemistry and immunohistochemistry showed that the inhibition of PG synthesis by beta-d-xyloside resulted in reduced incorporation of chondroitin sulfate into cartilage and bone matrix. This was associated with a 75% reduction in cell growth in condyle, determined by DNA synthesis, and in collagenous matrix synthesis in condyle and calvaria, evaluated by tritiated proline incorporation and type I collagen immunohistochemistry. Morphological and quantitative autoradiographic analyses also showed that inhibition of PG synthesis by beta-d-xyloside blocked bone matrix formation by perichondral progenitor cells in condyles and by osteoblasts in calvaria. In addition, alteration of PG metabolism blocked the mitogenic response to rhFGF-2 in calvaria. The data show that functional proteoglycans are essential for osteogenesis and for the growth response to FGF-2 during osteogenic differentiation in vitro.     

Could the effect of modeled microgravity on osteogenic differentiation of human mesenchymal stem cells be reversed by regulation of signaling pathways?

Microgravity (MG) results in a reduction in bone formation. Bone formation involves osteogenic differentiation from mesenchymal stem cells (hMSCs) in bone marrow. We modeled MG to determine its effects on osteogenesis of hMSCs and used activators or inhibitors of signaling factors to regulate osteogenic differentiation. Under osteogenic induction, MG reduced osteogenic differentiation of hMSCs and decreased the expression of osteoblast gene markers. The expression of Runx2 was also inhibited, whereas the expression of PPARgamma2 increased. MG also decreased phosphorylation of ERK, but increased phosphorylation of p38MAPK. SB203580, a p38MAPK inhibitor, was able to inhibit the phosphorylation of p38MAPK, but did not reduce the expression of PPARgamma2. Bone morphogenetic protein (BMP) increased the expression of Runx2. Fibroblast growth factor 2 (FGF2) increased the phosphorylation of ERK, but did not significantly increase the expression of osteoblast gene markers. The combination of BMP, FGF2 and SB203580 significantly reversed the effect of MG on osteogenic differentiation of hMSCs. Our results suggest that modeled MG inhibits the osteogenic differentiation and increases the adipogenic differentiation of hMSCs through different signaling pathways. Therefore, the effect of MG on the differentiation of hMSCs could be reversed by the mediation of signaling pathways.     

Self-maintenance of induced bone tissue]

The capacity for self-maintenance of the bone marrow osteogenic precursor cells from the skeletal bones and from the bones induced by implantation of decalcified bone matrix is compared. Transplantation in diffusion chambers is employed as the test system. Osteogenesis in the bone marrow transplants isolated from the skeletal bone lasts several months, whereas osteogenesis in the bone marrow transplants isolated from induced bone stops after the second month. Fibroblasts arising in the monolayer cultures of the skeletal bone marrow retained their osteogenic potencies after repeated passages. On the contrary, fibroblasts from the monolayer cultures of induced bone marrow lost their osteogenic capacity after the second passage. Thus, contrary to osteogenic precursors of the skeletal bone, osteogenic precursors of induced bone tissue had a very limited self-maintaining capacity after the cessation of induction.     

Osteogenic cell lineage analysis is facilitated by organ cultures of embryonic chick periosteum

Monoclonal antibodies against the surface of embryonic osteogenic cells (SB-1, SB-2, SB-3, and SB-5) have been used to characterize the sequence of transitions involved in the osteogenic cell lineage. In the present study, immunohistochemical analyses of the expression of osteogenic cell surface antigens in organ cultures of folded chick periosteum were performed. Unlike traditional culture methods using isolated osteoblastic cells, periosteal explants form a mineralized bone tissue in 4 to 6 days which is virtually identical to the in vivo counterpart. Examination of fresh explants confirm that no mature osteoblastic cells were present, although a discontinuous layer of preosteoblasts was evident. As the wave of osteodifferentiation swept through the cultured tissue, antibody SB-1 reacted with the surface of a large family of cells associated with the developing bone. Antibodies SB-3 and SB-2 reacted with progressively smaller subsets of cells, namely those in successively closer physical association with the newly formed and mineralizing bone. Cells recently encased in bone matrix were stained by both SB-2 and SB-5 antibodies, while those cells deep within the matrix reacted only with antibody SB-5. Analysis of this culture model allows dissection of the discrete cellular transition steps of osteogenesis, and reveals that osteogenic precursor cells proceed through the unique lineage stages which have been documented for in vivo osteogenesis. This culture system has furthermore provided evidence which is used to refine our understanding of the osteogenic cell lineage.     

Computer modelling of bone’s adaptation: the role of normal strain,shear strain and fluid flow

Bone loss is a serious health problem. In vivo studies have found that mechanical stimulation may inhibit bone loss as elevated strain in bone induces osteogenesis, i.e. new bone formation. However, the exact relationship between mechanical environment and osteogenesis is less clear. Normal strain is considered as a prime stimulus of osteogenic activity; however, there are some instances in the literature where osteogenesis is observed in the vicinity of minimal normal strain, specifically near the neutral axis of bending in long bones. It suggests that osteogenesis may also be induced by other or secondary components of mechanical environment such as shear strain or canalicular fluid flow. As it is evident from the literature, shear strain and fluid flow can be potent stimuli of osteogenesis. This study presents a computational model to investigate the roles of these stimuli in bone adaptation. The model assumes that bone formation rate is roughly proportional to the normal, shear and fluid shear strain energy density above their osteogenic thresholds. In vivo osteogenesis due to cyclic cantilever bending of a murine tibia has been simulated. The model predicts results close to experimental findings when normal strain, and shear strain or fluid shear were combined. This study also gives a new perspective on the relation between osteogenic potential of micro-level fluid shear and that of macro-level bending shear. Attempts to establish such relations among the components of mechanical environment and corresponding osteogenesis may ultimately aid in the development of effective approaches to mitigating bone loss.     

Induction of bone formation by recombinant human osteogenic protein-1 and sintered porous hydroxyapatite in adult primates

A critical issue in tissue engineering and morphogenesis of bone is the development of novel biomimetic biomaterials that are capable of optimizing the biological activity of recombinant human bone morphogenetic and osteogenic proteins, which are molecules that initiate bone formation in vivo. From a therapeutic perspective, a carrier matrix is required for the local delivery of these proteins to evoke a desired osteogenic effect. In view of the affinity of these proteins for hydroxyapatite, which may reflect the in vivo supramolecular assembly of bone proteins bound to both the extracellular matrix and the mineral component of bone, we investigated the efficacy of single applications of different doses of human osteogenic protein-1 (hOP-1) adsorbed onto sintered porous hydroxyapatites for bone induction in orthotopic calvarial defects in 12 adult male baboons (Papio ursinus) and heterotopically in the rectus abdominis of four additional baboons. In orthotopic specimens, pretreatment of sintered porous hydroxyapatites with 100 microgram of hOP-1 in 500 microliter of 5 mM hydrochloric acid resulted in rapid and diffuse osteoinduction restricted within the porous spaces of the hydroxyapatite, as evaluated by histology and histomorphometry on day 30. Hydroxyapatites treated with 500 microgram of hOP-1 showed a different pattern of bone formation and distribution on day 30 as compared with the lower dose of the recombinant morphogen. Although bone formation was extensive with the higher dose, it was found on the endocranial and pericranial aspects of the specimens, enveloping the implanted hydroxyapatite carrier, and the internal porous spaces were occupied by a rich vascular network without any bone formation. By 90 and 365 days after the implantation of both doses of hOP-1, however, there was remodelling and complete penetration of the newly induced bone within the available porous spaces. The combination of hOP-1 and hydroxyapatite also showed extensive bone formation in heterotopic specimens harvested from the rectus abdominis muscle of the baboon using doses of 5, 25, and 45 microgram of hOP-1 per implant. These findings in the adult primate demonstrate extensive bone formation by hOP-1 adsorbed onto sintered porous hydroxyapatites and suggest that predictable osteogenesis in clinical contexts for treatment of craniofacial bone defects may be engineered using inorganic, nonimmunogenic, and carvable delivery systems that initiate osteogenesis with relatively low doses of recombinant osteogenic proteins, thus mimicking the macrostructure and microstructure of living bone.     

Vibration loading promotes osteogenic differentiation of bone marrow-derived mesenchymal stem cells via p38 MAPK signaling pathway

Low magnitude high frequency vibration (LMHFV) exhibits effectively anabolic effects on the bone tissue, and can promote osteogenic differentiation of mesenchymal stem cells (MSCs) in vitro. The role of p38 MAPK signaling in LMHFV-induced osteogenesis remains unclear. In this current study, LMHFV loading was applied to BMSCs in vitro, and cell proliferation, alkaline phosphatase (ALP), matrix mineralization, as well as osteogenic genes expression were assayed. The mechanism of mechanical signal transduction was analysed using PCR array, qRT-PCR and Western blot. LMHFV increased cell proliferation in the growth medium, while inhibited proliferation in the osteogenic medium. ALP activity, matrix mineralization and osteogenic genes expression of Runx2, Col-I, ALP, OPN and OC were increased by LMHFV. p38 and MKK6 genes expression, and p38 phosphorylation were promoted in LMHFV-induced osteogenesis. Inhibition of p38 MAPK with SB203580 and targeted p38 siRNA blunted the increased ALP activity and osteogenic genes expression by LMHFV. These findings suggest that LMHFV promotes osteogenic differentiation of BMSCs, and p38 MAPK signaling shows an important function in LMHFV-induced osteogenesis.     

Development of osteogenic tissue in diffusion chambers from early precursor cells in bone marrow of adult rats

Diffusion chambers containing bone marrow cells from adult rats were implanted intraperitoneally into rat hosts and cultured in vivo for up to 64 days. Biochemical and histological analyses of the contents of the chambers demonstrate that a connective tissue consisting of bone, cartilage and fibrous tissues is formed by precursor cells present in marrow stroma. The amounts of osteogenic tissue and DNA are directly correlated with time of implantation and with number of cells inoculated. In the chambers there is initial formation of fibrous tissue which is strongly reactive to collagen type III, laminin and fibronectin. In areas of osteogenesis which appear later within this fibrous anlage, expression of collagen type III, laminin and fibronectin decrease and collagen types I and II increase in association with bone and cartilage respectively. Where osteogenesis does not develop, fibrous tissue continues to express collagen type III. The sequential expression of the different extracellular matrix components is similar to that previously observed during osteogenic differentiation in embryonic and adult developmental systems. It is concluded that the formation of fibrous and osteogenic tissues in diffusion chambers by precursor cells present in adult marrow, resembles the normal developmental process.     

Effects of high-dose estrogen on murine hematopoietic bone marrow precede those on osteogenesis

High-dose estrogen both stimulates new medullary bone formation and suppresses hematopoiesis in mouse long bones. To determine whether the latter response is a direct consequence of the former, we compared the time course of estrogen's effects on osteogenesis and hematopoietic bone marrow. Flow cytometry was employed to measure hematopoietic subpopulations in bone marrow from femurs of female mice killed at different times after commencing 0.5 mg estradiol/wk to each animal. Estrogen markedly reduced the number of leucocytes (CD11a positive), which had already diminished by 75% after 4 days and had virtually disappeared by 18 days. Specific populations showed a similar pattern of decline after estrogen, including B lymphocytes, monocytes, and endothelial cells. In contrast, the osteogenic precursor population showed a marked increase after estrogen treatment, as assessed by assaying alkaline phosphatase-positive colony-forming units (fibroblastic) ex vivo. However, this rise did not reach significance until 8 days after estrogen administration, suggesting that it follows rather than precedes estrogen's effects on hematopoiesis. We conclude that estrogen does not suppress hematopoiesis in mouse long bones as a direct consequence of its effects on osteogenesis.     

Enhancement of osteogenic differentiation of adipose-derived stem cells by PRP modified nanofibrous scaffold

Recent developments in bone tissue engineering have paved the way for more efficient and cost-effective strategies. Additionally, utilization of autologous sources has been considered very desirable and is increasingly growing. Recently, activated platelet rich plasma (PRP) has been widely used in the field of bone tissue engineering, since it harbours a huge number of growth factors that can enhance osteogenesis and bone regeneration. In the present study, the osteogenic effects of PRP coated nanofibrous PES/PVA scaffolds on adipose-derived mesenchymal stem cells have been investigated. Common osteogenic markers were assayed by real time PCR. Alkaline phosphate activity, calcium deposition and Alizarin red staining assays were performed as well. The results revealed that the highest osteogenic differentiation occurred when cells were cultured on PRP coated PES/PVA scaffolds. Interestingly, direct application of PRP to culture media had no additive effects on osteogenesis of cells cultured on PRP coated PES/PVA scaffolds or those receiving typical osteogenic factors. The highest osteogenic effects were achieved by the simplest and most cost-effective method, i.e. merely by using PRP coated scaffolds. PRP coated PES/PVA scaffolds can maximally induce osteogenesis with no need for extrinsic factors. The major contribution of this paper to the current researches on bone regeneration is to suggest an easy, cost-effective approach to enhance osteogenesis via PRP coated scaffolds, with no additional external growth factors.     

Calcium phosphate surfaces promote osteogenic differentiation of mesenchymal stem cells

Although studies in vivo revealed promising results in bone regeneration after implantation of scaffolds together with osteogenic progenitor cells, basic questions remain how material surfaces control the biology of mesenchymal stem cells (MSC). We used human MSC derived from bone marrow and studied the osteogenic differentiation on calcium phosphate surfaces. In osteogenic differentiation medium MSC differentiated to osteoblasts on hydroxyapatite and BONITmatrix, a degradable xerogel composite, within 14 days. Cells revealed a higher alkaline phosphatase (ALP) activity and increased RNA expression of collagen I and osteocalcin using real-time RTPCR compared with cells on tissue culture plastic. To test whether material surface characteristics alone are able to stimulate osteogenic differentiation, MSC were cultured on the materials in expansion medium without soluble additives for osteogenic differentiation. Indeed, cells on calcium phosphate without osteogenic differentiation additives developed to osteoblasts as shown by increased ALP activity and expression of osteogenic genes, which was not the case on tissue culture plastic. Because we reasoned that the stimulating effect on osteogenesis by calcium phosphate surfaces depends on an altered cell-extracellular matrix interaction we studied the dynamic behaviour of focal adhesions using cells transfected with GFP labelled vinculin. On BONITmatrix, an increased mobility of focal adhesions was observed compared with cells on tissue culture plastic. In conclusion, calcium phosphate surfaces are able to drive MSC to osteoblasts in the absence of osteogenic differentiation supplements in the medium. An altered dynamic behaviour of focal adhesions on calcium phosphate surfaces might be involved in the molecular mechanisms which promote osteogenic differentiation.     

Osteogenic differentiation of human adipose tissue-derived stromal cells (hASCs) in a porous three-dimensional scaffold

Recent studies have shown that liposuction aspirates from rat, rabbit, mouse, and human sources contain pluripotent adipose tissue-derived stromal cells (ASCs) that can differentiate into various mesodermal cell types, including osteoblasts, myoblasts, chondroblasts, and preadipocytes. To develop a research model for autologous bone tissue engineering, we isolated ASCs from human liposuction aspirates (hASCs) and induced their osteogenic differentiation in three-dimensional poly(dl-lactic-co-glycolic acid) (PLGA) scaffolds. Human liposuction aspirates were proteolytically digested and centrifuged to obtain hASCs. After primary culture in control media and expansion to three passages, the cells were seeded in two-dimensional plates or three-dimensional PLGA scaffolds and cultured in osteogenic media for 4 weeks. In two-dimensional culture, osteogenesis was assessed by RT-PCR analysis of the osteogenic-specific bone sialoprotein mRNA, by alkaline phosphatase staining, and by von Kossa staining. In three-dimensional culture, osteogenesis was assessed by von Kossa and alizarine red S staining at 1, 2, and 4 weeks following osteogenic induction. hASCs incubated in two-dimensional osteogenic media stained positively for alkaline phosphatase and with von Kossa stain after 2 weeks of differentiation. Expression of the osteogenesis-specific bone sialoprotein gene was detected by RT-PCR after 2 weeks of differentiation. PLGA scaffolds seeded with hASCs showed multiple calcified extracellular matrix nodules by von Kossa and alizarine red S staining after 2 weeks of differentiation. In conclusion, the authors identified an osteogenic potential of hASCs and demonstrated osteogenic differentiation of hASCs into an osteogenic lineage in three-dimensional PLGA scaffolds.     

Gentiopicroside promotes the osteogenesis of bone mesenchymal stem cells by modulation of β-catenin-BMP2 signalling pathway

Osteoporosis is characterized by increased bone fragility, and the drugs used at present to treat osteoporosis can cause adverse reactions. Gentiopicroside (GEN), a class of natural compounds with numerous biological activities such as anti-resorptive properties and protective effects against bone loss. Therefore, the aim of this work was to explore the effect of GEN on bone mesenchymal stem cells (BMSCs) osteogenesis for a potential osteoporosis therapy. In vitro, BMSCs were exposed to GEN at different doses for 2 weeks, whereas in vivo, ovariectomized osteoporosis was established in mice and the therapeutic effect of GEN was evaluated for 3 months. Our results in vitro showed that GEN promoted the activity of alkaline phosphatase, increased the calcified nodules in BMSCs and up-regulated the osteogenic factors (Runx2, OSX, OCN, OPN and BMP2). In vivo, GEN promoted the expression of Runx2, OCN and BMP2, increased the level of osteogenic parameters, and accelerated the osteogenesis of BMSCs by activating the BMP pathway and Wnt/β-catenin pathway, effect that was inhibited using the BMP inhibitor Noggin and Wnt/β-catenin inhibitor DKK1. Silencing the β-catenin gene and BMP2 gene blocked the osteogenic differentiation induced by GEN in BMSCs. This block was also observed when only β-catenin was silenced, although the knockout of BMP2 did not affect β-catenin expression induced by GEN. Therefore, GEN promotes BMSC osteogenesis by regulating β-catenin-BMP signalling, providing a novel strategy in the treatment of osteoporosis.