Biomolecular mechanisms of calvarial bone induction: immature versus mature dura mater

The ability of newborns and immature animals to reossify calvarial defects has been well described. This capacity is generally lost in children greater than 2 years of age and in mature animals. The dura mater has been implicated as a regulator of calvarial reossification. To date, however, few studies have attempted to identify biomolecular differences in the dura mater that enable immature, but not mature, dura to induce osteogenesis. The purpose of these studies was to analyze metabolic characteristics, protein/gene expression, and capacity to form mineralized bone nodules of cells derived from immature and mature dura mater. Transforming growth factor beta-1, basic fibroblast growth factor, collagen type IalphaI, osteocalcin, and alkaline phosphatase are critical growth factors and extracellular matrix proteins essential for successful osteogenesis. In this study, we have characterized the proliferation rates of immature (6-day-old rats, n = 40) and mature (adult rats, n = 10) dura cell cultures. In addition, we analyzed the expression of transforming growth factor beta-1, basic fibroblast growth factor-2, proliferating cell nuclear antigen, and alkaline phosphatase. Our in vitro findings were corroborated with Northern blot analysis of mRNA expression in total cellular RNA isolated from snap-frozen age-matched dural tissues (6-day-old rats, n = 60; adult rats, n = 10). Finally, the capacity of cultured dural cells to form mineralized bone nodules was assessed. We demonstrated that immature dural cells proliferate significantly faster and produce significantly more proliferating cell nuclear antigen than mature dural cells (p < 0.01). Additionally, immature dural cells produce significantly greater amounts of transforming growth factor beta-1, basic fibroblast growth factor-2, and alkaline phosphatase (p < 0.01). Furthermore, Northern blot analysis of RNA isolated from immature and mature dural tissues demonstrated a greater than 9-fold, 8-fold, and 21-fold increase in transforming growth factor beta-1, osteocalcin, and collagen IalphaI gene expression, respectively, in immature as compared with mature dura mater. Finally, in keeping with their in vivo phenotype, immature dural cells formed large calcified bone nodules in vitro, whereas mature dural cells failed to form bone nodules even with extended culture. These studies suggest that differential expression of growth factors and extracellular matrix molecules may be a critical difference between the osteoinductive capacity of immature and mature dura mater. Finally, we believe that the biomolecular bone- and matrix-inducing phenotype of immature dura mater regulates the ability of young children and immature animals to heal calvarial defects.     

Co-culture of osteoblasts with immature dural cells causes an increased rate and degree of osteoblast differentiation

For decades surgeons have exploited the ability of infants to reossify large calvarial defects. To demonstrate the role of dura mater-osteoblast communication during the process of calvarial reossification, the authors used a novel in vitro system that recapitulates the in vivo anatomic relationship of these cell populations. Primary cultures of osteoblast cells from 2-day-old Sprague-Dawley rat pups were grown on six-well plates, and cultures of immature, non-suture-associated dura mater cells from 6-day-old Sprague-Dawley rat pups were grown on Transwell inserts. When the osteoblast and dura mater cell cultures reached confluence, they were combined. This Transwell co-culture system permitted the two cell populations to grow together in the same well, but it prevented direct cell-to-cell contact. Therefore, the authors were able to determine, for the first time, whether paracrine signaling from immature, non-suture-associated dura mater could influence the biologic activity of osteoblasts.Osteoblasts co-cultured with dural cells proliferated significantly faster after 2 days (2.1 x 10(5) +/- 2.4 x 10(4) versus 1.4 x 10(5) +/- 2.2 x 10(4), p < or = 0.05) and 4 days (3.1 x 10(5) +/- 5 x 10(4) versus 2.2 x 10(5) +/- 4.0 x 10(4), p < or = 0.01) than did osteoblasts cultured alone. After 20 days, co-cultured osteoblasts expressed greater amounts of mRNA for several markers of osteoblast differentiation, including collagen I alpha I (4-fold), alkaline phosphatase (2.5-fold), osteopontin (3-fold), and osteocalcin (4-fold), than did osteoblasts cultured alone. After 30 days, co-cultured osteoblasts produced bone nodules that were significantly greater both in number (324 +/- 29 nodules versus 252 +/- 29 nodules per well, p , < or = 0.04) and total area of nodules (65 +/- 11 mm(2) versus 24 +/- 1.6 mm(2), p < or = 0.003) than osteoblasts cultured alone.To begin to understand how dural cells effect changes in osteoblast gene expression, the authors compared the expression of candidate genes, transforming growth factor beta 1 and fibroblast growth factor 2, in dural cells and osteoblasts before and after 5 days of culture. Interestingly, the dura mater produced marked amounts of these osteogenic cytokines compared with osteoblasts.The described co-culture system demonstrated that co-cultured osteoblasts proliferated more rapidly and experienced an increased rate and degree of cellular maturation than did osteoblasts cultured alone. The authors hypothesize that this effect was due to paracrine signaling (e.g., transforming growth factor beta 1 and fibroblast growth factor 2) from the dura mater, and they are investigating those mechanisms in ongoing experiments. Collectively these data verify that immature, non-suture-associated dura mater can influence the biologic activity of osteoblasts. Moreover, the production of cytokines derived from the dura mater (e.g., transforming growth factor beta 1 and fibroblast growth factor 2), and they may begin to explain why immature animals and infants with intact dura mater can reossify large calvarial defects.     

Mechanical strain affects dura mater biological processes: implications for immature calvarial healing

The human brain grows rapidly during the first 2 years of life. This growth generates tensile strain in the overlying dura mater and neurocranium. Interestingly, it is largely during this 2-year growth period that infants are able to reossify calvarial defects. This clinical observation is important because it suggests that calvarial healing is most robust during the period of active intracranial volume expansion. With a rat model, it was previously demonstrated that immature dura mater proliferates more rapidly and produces more osteogenic cytokines and markers of osteoblast differentiation than does mature dura mater. It was therefore hypothesized that mechanical strain generated by the growing brain induces immature dura mater proliferation and increases osteogenic cytokine expression necessary for growth and healing of the overlying calvaria. Human and rat (n = 40) intracranial volume expansion was calculated as a function of age. These calculations demonstrated that 83 percent of human intracranial volume expansion is complete by 2 years of age and 90 percent of Sprague-Dawley rat intracranial volume expansion is achieved by 2 months of age. Next, the maximal daily circumferential tensile strains that could be generated in immature rat dura mater were calculated, and the corresponding daily biaxial tensile strains in the dura mater during this 2-month period were determined. With the use of a three-parameter monomolecular growth curve, it was calculated that rat dura mater experiences daily equibiaxial strains of at most 9.7 percent and 0.1 percent at birth (day 0) and 60 days of age, respectively. Because it was noted that immature dural cells may experience tensile strains as high as approximately 10 percent, neonatal rat dural cells were subjected to 10 percent equibiaxial strain in vitro, and dural cell proliferation and gene expression profiles were analyzed. When exposed to mechanical strain, immature dural cells rapidly proliferated (5.8-fold increase in proliferating cell nuclear antigen expression at 24 hours). Moreover, mechanical strain induced marked up-regulation of dural cell osteogenic cytokine production; transforming growth factor-beta1 messenger RNA levels increased 3.4-fold at 3 hours and fibroblast growth factor-2 protein levels increased 4.5-fold at 24 hours and 5.6-fold at 48 hours. Finally, mechanical strain increased dural cell expression of markers of osteoblast differentiation (2.8-fold increase in osteopontin levels at 3 hours). These findings suggest that mechanical strain can induce changes in dura mater biological processes and gene expression that may play important roles in coordinating the growth and healing of the neonatal calvaria.     

Osteogenesis in calvarial defects: contribution of the dura,the pericranium,and the surrounding bone in adult versus infant animals

Guided bone regeneration is a promising means for reconstructing bone defects in the cranium. The present study was performed to better define those factors that affect osteogenesis in the cranium. The authors studied a single animal model, investigating the contribution of the dura, the pericranium, and the adjacent calvarial bone in the process of calvarial regeneration in both mature and immature animals. Bilateral, 100-mm2, parietal calvariectomies were performed in immature (n = 16) and mature (n = 16) rabbits. Parietal defects were randomized to one of four groups depending on the differential blockade of the dura and/or the pericranium by expanded polytetrafluoroethylene membranes. Animals were humanely killed after 12 weeks, and histometric analysis was performed to quantitate the area of the original bone defect, new bone formation, and new bone density. Bone formation was quantified separately both at the periphery and in the center of the defects. Extrasite bone formation was also quantified both on the dural and on the pericranial sides of the barriers. Bone regeneration was incomplete in all groups over the 12-week study period, indicating that complete bone healing was not observed in any group. The dura was more osteogenic than the pericranium in mature and immature animals, as there was significantly more extrasite bone formed on the dural side in the double expanded polytetrafluoroethylene barrier groups. In both the dural and the double expanded polytetrafluoroethylene barrier groups, dural bone production was significantly greater in immature compared with mature animals. The dura appeared to be the source of central new bone, because dural blockade in the dural and double expanded polytetrafluoroethylene groups resulted in a significant decrease in central bone density in both mature and immature animals. Paradoxically, isolation of the pericranium in mature animals resulted in a significant reduction in total new bone area, whereas pericranial contact appeared to enhance peripheral new bone formation, with the control group having the greatest total new bone area. The present study establishes a model to quantitatively study the process of bone regeneration in calvarial defects and highlights differences in the contribution of the dura and pericranium to calvarial bone regeneration between infant and adult animals. On the basis of these findings, the authors propose that subsequent studies in which permeability of the expanded polytetrafluoroethylene membranes is altered to permit migration of osteoinductive proteins into the defect while blocking prolapse of adjacent soft tissues may help to make guided bone regeneration a realistic alternative for the repair of cranial defects.     

Dura mater-derived FGF-2 mediates mitogenic signaling in calvarial osteoblasts

Although dura mater tissue is believed to have an important role in calvarial reossification in many in vivo studies, few studies have shown the direct effect of dura mater cells on osteoblasts. In addition, no reports have yet identified the potential factor(s) responsible for various biological activities exerted by dura mater on calvarial reossification (e.g., cell proliferation). In this study, we tested the effect of dura mater on calvarial-derived osteoblasts by performing both heterotypic coculture and by culturing osteoblast cells with conditioned media harvested from dura mater cells of juvenile (3-day-old) and adult (30-day-old) mice. The results presented here demonstrate that cellular proliferation of juvenile osteoblast cells was significantly increased by juvenile dura mater either in the coculture system or when dura mater cell-conditioned medium was applied to the osteoblast cells. Moreover, high levels of FGF-2 protein were detected in juvenile dura mater cells and their conditioned medium. In contrast, low levels of FGF-2 protein were detected in adult dura mater cells, whereas FGF-2 protein was not detectable in their conditioned medium. Abrogation of the mitogenic effect induced by juvenile dura mater cell-conditioned medium was achieved by introducing a neutralizing anti-FGF-2 antibody, thus indicating that FGF-2 may be responsible for the mitogenic effect of the juvenile dura mater. Moreover, data obtained by exploring the three major FGF-2 signaling pathways further reinforced the idea that FGF-2 might be an important paracrine signaling factor in vivo supplied by the underlying dura mater to the overlying calvarial osteoblasts.     

Dura mater biology: autocrine and paracrine effects of fibroblast growth factor 2

The dura mater, the outermost layer of the meninges, is thought to be essential for calvarial morphogenesis, postnatal suture fusion, and osseous repair of calvarial defects. Despite numerous studies illustrating the fundamental role of the dura mater, there is little information about the autocrine and paracrine mechanisms regulating dural cell biology during calvarial ossification. Previous work conducted in the authors' laboratory demonstrated that non-suture-associated dural cells from 6-day-old rat pups expressed high levels of fibroblast growth factor 2 (FGF-2), whereas dural cells from 60-day-old adult rats expressed very little FGF-2. Because young mammals can successfully heal large calvarial defects, the authors sought to investigate the autocrine and/or paracrine effects of FGF-2 on the proliferation, gene expression, and alkaline phosphatase production of dural cells.Cultures of non-suture-associated dural cells were established from 6-day-old Sprague-Dawley rat pups and then stimulated with recombinant human FGF-2 (rhFGF-2; 10 ng/ml). Dural cells stimulated with rhFGF-2 proliferated significantly faster than untreated dural cells at 24 hours (2.1 x 10(5) +/- 3.2 x 10(4) versus 1.1 x 10(5) +/- 1.8 x 10(4), p < or = 0.001) and 48 hours (2.3 x 10(5) +/- 4.2 x 10(4) versus 1.2 x 10(5) +/- 1.3 x 10(4), p < or = 0.001). Moreover, dural cells stimulated with rhFGF-2 expressed 7-fold more proliferating cell nuclear antigen than did control cultures. Treatment with rhFGF-2 increased dural cell expression of genes important for skeletal repair: FGF-2 (7-fold), transforming growth factor beta 1 (3-fold), transforming growth factor beta 3 (4-fold), and type I collagen (4-fold). Furthermore, rhFGF-2 increased dural cell expression of osteopontin (2-fold), a "late" marker of osteoblastic differentiation. Interestingly, dural cell alkaline phosphatase activity, an "earlier" marker of osteoblast differentiation, was significantly decreased by treatment with rhFGF-2 compared with control cultures at 24 hours (0.005 +/- 0.001 versus 0.01 +/- 0.003, p < or = 0.01) and 48 hours (0.004 +/- 0.0009 versus 0.01 +/- 0.0009). Together these data provide insight into the autocrine and paracrine effects of FGF-2 on the biology of the dura mater.     

Basic fibroblast growth factor and transforming growth factor beta-1 expression in the developing dura mater correlates with calvarial bone formation

Numerous studies have found dura mater-calvarial mesenchyme interactions during calvarial bone induction; however, the exact molecular mechanisms governing these inductive events remain unknown. Recent studies have implicated basic fibroblast growth factor (FGF-2) and transforming growth factor-beta1 (TGF-beta1) in regulating bone formation. The purpose of this study was, therefore, to investigate the expression of FGF-2 and TGF-beta1 during calvarial bone formation in rats. Eight rats were killed on embryonic days 14, 18, and 20 and neonatal day 1 (n = 32). Four animals at each time point were analyzed by in situ hybridization, and the remainder were analyzed by immunohistochemistry. The results indicated that the dura mater underlying the developing calvarial bone strongly expressed FGF-2 and TGF-beta1 mRNA at all time points examined. In contrast, minimal growth factor expression was noted in the overlying calvarial mesenchyme until embryonic day 18, but it increased significantly with increasing age. Importantly, FGF-2 and TGF-beta1 mRNA expression in the dura mater underlying the developing calvarium preceded and was significantly greater than expression in the calvarium mesenchyme (p < 0.05). Interestingly, minimal expression of FGF-2 and TGF-beta1 mRNA was noted for all time points in the dura mater underlying the posterior frontal suture and within the posterior frontal suture connective tissue (p < 0.01 when compared with the dura mater underlying the developing calvarium). Immunohistochemical findings closely paralleled mRNA expression, with intense staining for FGF-2 and TGF-beta1 in the dura mater underlying the developing calvarial mesenchyme. Increasing FGF-2 and TGF-beta1 staining was noted within calvarial osteoblasts with increasing age, particularly in cells located near the endocranial surface (i.e., in contact with the developing dura mater). These findings, together with the known biologic functions of FGF-2 and TGF-beta1, implicate these growth factors in the regulation of calvarial bone growth by the developing dura mater. The possible mechanisms of this interaction are discussed.     

Regional differentiation of rat cranial suture-derived dural cells is dependent on association with fusing and patent cranial sutures

A significant body of literature supports a role for the dura mater underlying cranial sutures in the regulation of sutural fate. These studies have implicated regional differentiation of the dura mater based on association with fusing and patent rat cranial sutures. The purpose of these experiments was to isolate and characterize dural cells associated with fusing (posterior frontal) and patent (sagittal) rat cranial sutures. Six-day-old rats were killed, and the dura mater underlying the posterior frontal and sagittal sutures was harvested. Dural cells were briefly trypsinized and allowed to reach confluence. Two litters (10 animals per litter) were used for each set of experiments. Cells were harvested after the first and fifth passages for analysis of vimentin and desmoplakin expression (characteristic of human meningeal cells), cellular proliferation, density at confluence (a measure of cellular contact inhibition), and alkaline phosphatase production. In addition, bone nodule formation and collagen I production were analyzed in first passage cells. The results indicate that suture-derived dural cells can be established and that these cells coexpress vimentin and desmoplakin. In addition, it is demonstrated that first-passage sagittal suture-derived dural cells proliferate significantly faster and have decreased cellular contact inhibition than posterior frontal suture-derived cells (p < 0.01). Finally, it is shown that suture-derived dural cells have osteoblast-like properties, including alkaline phosphatase production, collagen I expression, and bone nodule formation in vitro. The possible mechanisms by which regional differentiation of suture-derived dural cells occur are discussed.     

Differential growth factor adsorption to calvarial osteoblast-secreted extracellular matrices instructs osteoblastic behavior

Craniosynostosis (CS), the premature ossification of cranial sutures, is attributed to increased osteogenic potential of resident osteoblasts, yet the contribution of the surrounding extracellular matrix (ECM) on osteogenic differentiation is unclear. The osteoblast-secreted ECM provides binding sites for cellular adhesion and regulates the transport and signaling of osteoinductive factors secreted by the underlying dura mater. The binding affinity of each osteoinductive factor for the ECM may amplify or mute its relative effect, thus contributing to the rate of suture fusion. The purpose of this paper was to examine the role of ECM composition derived from calvarial osteoblasts on protein binding and its resultant effect on cell phenotype. We hypothesized that potent osteoinductive proteins present during sutural fusion (e.g., bone morphogenetic protein-2 (BMP-2) and transforming growth factor beta-1 (TGF-β1)) would exhibit distinct differences in binding when exposed to ECMs generated by human calvarial osteoblasts from unaffected control individuals (CI) or CS patients. Decellularized ECMs produced by osteoblasts from CI or CS patients were incubated in the presence of BMP-2 or TGF-β1, and the affinity of each protein was analyzed. The contribution of ECM composition to protein binding was interrogated by enzymatically modulating proteoglycan content within the ECM. BMP-2 had a similar binding affinity for each ECM, while TGF-β1 had a greater affinity for ECMs produced by osteoblasts from CI compared to CS patients. Enzymatic treatment of ECMs reduced protein binding. CS osteoblasts cultured on enzymatically-treated ECMs secreted by osteoblasts from CI patients in the presence of BMP-2 exhibited impaired osteogenic differentiation compared to cells on untreated ECMs. These data demonstrate the importance of protein binding to cell-secreted ECMs and confirm that protein-ECM interactions have an important role in directing osteoblastic differentiation of calvarial osteoblasts.     

Dopamine Receptors in the Human Dura Mater

Dopamine receptors (Dar) were studied as a component of the nervous dopaminergic system in the human dura mater. Dar were stained in several dural zones (vascular, perivascular, intervascular) in different regions (basal, calvarial, tentorial, occipital, frontal, parietal, temporal) of the cranial meninges. Specimens of human dura mater were harvested from autopsies of 10 elderly male subjects (age range, 60-75 years). Dar were labeled with specific (H3) markers, studied with radiobinding techniques (including liquid scintillation), stained for light microscope autoradiography, and measured by means of quantitative analysis of images. All results were evaluated with statistical analysis to identify significant results. More dural Dar were found in the basal region than in the calvarial one. Moreover, Dar are more abundant in the vascular and perivascular dural zone than in the intervascular one. The vascular distribution of Dar seemed to indicate that Dar play a role in the control of meningeal blood vessels. The location and distribution of D1 and D2 receptors in the human cranial dura mater confirmed the presence of a dopaminergic system, which could play an important role in controlling blood flow and/or other functions of meningeal membranes.     

Assessment of the effects on growth of porous hydroxyapatite granule cranioplasty in the immature guinea pig craniofacial skeleton

The immature guinea pig was used to study the effects on growth of porous granular hydroxyapatite used as an onlay cranioplasty and inlay cranioplasty to reconstruct full-thickness cranial defects in a growing craniofacial skeleton. Forty Hartley guinea pigs, 20 immature animals and 20 mature animals, were divided into four groups each containing five mature and five immature animals. The mature animals served as controls. Group I underwent elevation and replacement of the parietal periosteum. Group II underwent placement of hydroxyapatite between periosteum and parietal bone. Group III underwent elevation and replacement of autogenous bone flap after the formation of a 1 x 1-cm craniectomy defect in the parietal skull. Group IV underwent elevation of a 1 x 1-cm parietal craniectomy and reconstruction of the defect with hydroxyapatite granules placed between the dura and periosteum. Immature animals were killed at maturity at 3.5 months and mature animals were killed 2.5 months postoperatively. Macroscopic examination of the operative field, transverse and longitudinal cephalometric measurements, and histological sections encompassing the operative sites were compared. Macroscopically, all reconstructed operative sites were fully incorporated into the cranium. Histological staining of the sectioned operative site revealed no hydroxyapatite migration through the cranial bone or dura. No inflammatory or foreign body reaction was evident in the subcutaneous tissue, periosteum, or dura. No statistically significant cephalometric intergroup or intragroup differences were found at the conclusion of the study. The results of this study indicate that a granular porous form of hydroxyapatite may be used as an onlay or inlay cranioplasty in the immature guinea pig craniofacial skeleton without evidence of dural inflammation, granule migration, or growth restriction or retardation.     

Rat mandibular distraction osteogenesis: part III. Gradual distraction versus acute lengthening

Distraction osteogenesis is a well-established method of endogenous tissue engineering. This technique has significantly augmented our armamentarium of reconstructive craniofacial procedures. Although the histologic and ultrastructural changes associated with distraction osteogenesis have been extensively described, the molecular mechanisms governing successful membranous distraction remain unknown. Using an established rat model, the molecular differences between successful (i.e., osseous union with gradual distraction) and ineffective (i.e., fibrous union with acute lengthening) membranous bone lengthening was analyzed. Herein, the first insight into the molecular mechanisms of successful membranous bone distraction is provided. In addition, these data provide the foundation for future targeted therapeutic manipulations designed to improve osseous regeneration. Vertical mandibular osteotomies were created in 52 adult male Sprague-Dawley rats, and the animals were fitted with customized distraction devices. Twenty-six animals underwent immediate acute lengthening (3 mm; a length previously shown to result in fibrous union) and 26 animals were gradually distracted (after a 3-day latency period, animals were distracted 0.25 mm twice daily for 6 days; total = 3 mm). Four mandibular regenerates were harvested from each group for RNA analysis on 5, 7, 9, 23, and 37 days postoperatively (n = 40). Two mandibular regenerates were also harvested from each group and prepared for immunohistochemistry on postoperative days 5, 7, and 37 (n = 12). In addition to the 52 experimental animals, 4 control rats underwent sham operations (skin incision only) and mandibular RNA was immediately collected. Control and experimental specimens were analyzed for collagen I, osteocalcin, tissue inhibitor of metalloproteinase-1, and vascular endothelial growth factor mRNA and protein expression. In this study, marked elevation of critical extracellular matrix molecules (osteocalcin and collagen I) during the consolidation phase of gradual distraction compared with acute lengthening is demonstrated. In addition, the expression of an inhibitor of extracellular matrix turnover, tissue inhibitor of metalloproteinase-1, remained strikingly elevated in gradually distracted animals. Finally, this study demonstrated that neither gradual distraction nor acute lengthening appreciably alters vascular endothelial growth factor expression. These results suggest that gradual distraction osteogenesis promotes successful osseous bone repair by regulating the expression of bone-specific extracellular matrix molecules. In contrast, decreased production or increased turnover of bone scaffolding proteins (i.e., collagen) or regulators of mineralization (i.e., osteocalcin) may lead to fibrous union during acute lengthening.     

FGF-2对人骨髓间充质干细胞增殖和向成骨细胞分化的影响

探讨体外培养条件下,成纤维细胞生长因子-2（FGF-2）和地塞米松（Dex）对第7代人骨髓间充质干细胞（MSCs）增殖和向成骨细胞分化的作用以及两者联合使用的效应。MSCs经含FGF-2或／和Dex的培养液作用后,于不同时间采用MTT法测定细胞增殖情况;对硝基苯磷酸（pNPP）法测定碱性磷酸酶（ALP）活性;ELISA法测定骨钙蛋白（OC）含量;茜素红S染色法对沉积的钙盐进行染色。发现：（1）FGF-2组细胞的生长速度为对照组的1．31倍,Dex／FGF-2组细胞的生长速度为FGF-2组的1．12倍。（2）Dex组的ALP活性、OC含量和细胞外基质钙盐沉积分别为对照组的17．0倍、2．12倍和10．56倍,并能形成成熟的羟基磷灰石（HA）结晶和骨结节;FGF-2组的ALP活性比对照组降低了76．7％,虽然OC含量、钙盐沉积增加,但不能形成成熟的HA结晶和骨结节;FGF-2对Dex诱导的ALP活性增加和HA结晶形成有拮抗作用。由此证明：（1）FGF-2可促进MSCs的增殖,Dex对MSCs的增殖无明显作用;Dex能增强FGF-2对MSCs的促增殖效应。（2）Dex可使MSCs分化为成熟的成骨细胞,是一个有效的成骨细胞分化诱导剂;FGF-2可使MSCs分化为未成熟的成骨细胞;FGF-2拮抗Dex诱导MSCs分化为成熟的成骨细胞。     

Adipose-derived adult stromal cells heal critical-size mouse calvarial defects

In adults and children over two years of age, large cranial defects do not reossify successfully, posing a substantial biomedical burden. The osteogenic potential of bone marrow stromal (BMS) cells has been documented. This study investigates the in vivo osteogenic capability of adipose-derived adult stromal (ADAS) cells, BMS cells, calvarial-derived osteoblasts and dura mater cells to heal critical-size mouse calvarial defects. Implanted, apatite-coated, PLGA scaffolds seeded with ADAS or BMS cells produced significant intramembranous bone formation by 2 weeks and areas of complete bony bridging by 12 weeks as shown by X-ray analysis, histology and live micromolecular imaging. The contribution of implanted cells to new bone formation was 84-99% by chromosomal detection. These data show that ADAS cells heal critical-size skeletal defects without genetic manipulation or the addition of exogenous growth factors.     

TGF-beta2 stimulates cranial suture closure through activation of the Erk-MAPK pathway

Cranial sutures are important growth sites of the skull. During suture closure, the dura mater is one of the most important sources of various positive and negative regulatory signals. Previous results indicate that TGF-beta2 from dura mater strongly accelerates suture closure, however, its exact regulatory mechanism is still unclear. In this study, we confirmed that removal of dura mater in calvarial organ culture strongly accelerates sagittal suture closure and that this effect is further enhanced by TGF-beta2 treatment. TGF-beta2 stimulated cell proliferation in the MC3T3-E1 cell line. Similarly, it stimulated the proliferation of cells in the sutural space in calvarial organ culture. Furthermore, TGF-beta2-mediated enhanced cell proliferation and suture closure were almost completely inhibited by an Erk-MAPK blocker, PD98059. These results indicate that TGF-beta2-induced activation of Erk-MAPK is an important signaling component that stimulates cell proliferation to enrich osteoprogenitor cells, thereby promoting their differentiation into osteoblasts to achieve a rapid calvarial bone expansion.     

Gene expression of TGF-beta, TGF-beta receptor, and extracellular matrix proteins during membranous bone healing in rats

Poorly healing mandibular fractures and osteotomies can be troublesome complications of craniomaxillofacial trauma and reconstructive surgery. Gene therapy may offer ways of enhancing bone formation by altering the expression of desired growth factors and extracellular matrix molecules. The elucidation of suitable candidate genes for therapeutic intervention necessitates investigation of the endogenously expressed patterns of growth factors during normal (i.e., successful) fracture repair. Transforming growth factor beta1 (TGF-beta1), its receptor (Tbeta-RII), and the extracellular matrix proteins osteocalcin and type I collagen are thought to be important in long-bone (endochondral) formation, fracture healing, and osteoblast proliferation. However, the spatial and temporal expression patterns of these molecules during membranous bone repair remain unknown. In this study, 24 adult rats underwent mandibular osteotomy with rigid external fixation. In addition, four identically treated rats that underwent sham operation (i.e., no osteotomy) were used as controls. Four experimental animals were then killed at each time point (3, 5, 7, 9, 23, and 37 days after the procedure) to examine gene expression of TGF-beta1 and Tbeta-RII, osteocalcin, and type I collagen. Northern blot analysis was used to compare gene expression of these molecules in experimental animals with that in control animals (i.e., nonosteotomized; n = 4). In addition, TGF-beta1 and T-RII proteins were immunolocalized in an additional group of nine animals killed on postoperative days 3, 7, and 37. The results of Northern blot analysis demonstrated a moderate increase (1.7 times) in TGF-beta1 expression 7 days postoperatively; TGF-beta1 expression returned thereafter to near baseline levels. Tbeta-RII mRNA expression was downregulated shortly after osteotomy but then increased, reaching a peak of 1.8 times the baseline level on postoperative day 9. Osteocalcin mRNA expression was dramatically downregulated shortly after osteotomy and remained low during the early phases of fracture repair. Osteocalcin expression trended slowly upward as healing continued, reaching peak expression by day 37 (1.7 times the control level). In contrast, collagen type IalphaI mRNA expression was acutely downregulated shortly after osteotomy, peaked on postoperative days 5, and then decreased at later time points. Histologic samples from animals killed 3 days after osteotomy demonstrated TGF-beta1 protein localized to inflammatory cells and extracellular matrix within the fracture gap, periosteum, and peripheral soft tissues. On postoperative day 7, TGF-beta1 staining was predominantly localized to the osteotomized bone edges, periosteum, surrounding soft tissues, and residual inflammatory cells. By postoperative day 37, complete bony healing was observed, and TGF-beta1 staining was localized to the newly formed bone matrix and areas of remodeling. On postoperative day 3, Tbeta-RII immunostaining localized to inflammatory cells within the fracture gap, periosteal cells, and surrounding soft tissues. By day 7, Tbeta-RII staining localized to osteoblasts of the fracture gap but was most intense within osteoblasts and mesenchymal cells of the osteotomized bone edges. On postoperative day 37, Tbeta-RII protein was seen in osteocytes, osteoblasts, and the newly formed periosteum in the remodeling bone. These observations agree with those of previous in vivo studies of endochondral bone formation, growth, and healing. In addition, these results implicate TGF-beta1 biological activity in the regulation of osteoblast migration, differentiation, and proliferation during mandibular fracture repair. Furthermore, comparison of these data with gene expression during mandibular distraction osteogenesis may provide useful insights into the treatment of poorly healing fractures because distraction osteogenesis has been shown to be effective in the management of these difficult clinical cases.