Measuring three-dimensional strain distribution in tendon.

Tendons are tough fibrous tissues that facilitate skeletal movement by transferring muscular force to bone. Studies into the effects of mechanical stress on tendons have shown that these can either accelerate healing or cause tendon injuries depending on the load applied. It is known that local strain magnitude and direction play an important role in tendon remodelling and also failure, and different techniques to study strain distribution have been proposed. Image registration and processing techniques are among the recently employed methods. In this study, a novel three-dimensional image processing technique using the Sheffield Image Registration Toolkit is introduced to study local strain and displacement distribution in tendon. The results show that the local normal strain values in the loading axis are smaller than the global applied load, and fibre sliding was detected as a dominant mechanism for transferring the applied load within tendon. However, results from different samples suggest three distinct modes of deformation during loading, as some show only parallel sliding of fibres in respect to the loading axis, whereas others are twisted or deflected in directions transverse to the loading axis. The proposed 3D image registration method is essential for analysing this out-of-plane movement, which cannot be detected using a standard 2D method.

Effect of age and diabetes on the response of mesenchymal progenitor cells to fibrin matrices.

Mesenchymal stem cells are showing increasing promise in applications such as tissue engineering and cell therapy. MSC are low in number in bone marrow, and therefore in vitro expansion is often necessary. In vivo, stem cells often reside within a niche acting to protect the cells. These niches are composed of niche cells, stem cells, and extracellular matrix. When blood vessels are damaged, a fibrin clot forms as part of the wound healing response. The clot constitutes a form of stem cell niche as it appears to maintain the stem cell phenotype while supporting MSC proliferation and differentiation during healing. This is particularly appropriate as fibrin is increasingly being suggested as a scaffold meaning that fibrin-based tissue engineering may to some extent recapitulate wound healing. Here, we describe how fibrin modulates the clonogenic capacity of MSC derived from young/old human donors and normal/diabetic rats. Fibrin was prepared using different concentrations to modulate the stiffness of the substrate. MSC were expanded on these scaffolds and analysed. MSC showed an increased self-renewal on soft surfaces. Old and diabetic cells lost the ability to react to these signals and can no longer adapt to the changed environment.

Short bouts of mechanical loading are as effective as dexamethasone at inducing matrix production by human bone marrow mesenchymal stem cell.

Dexamethasone (Dex) is used widely to induce differentiation in human mesenchymal stem cells (hMSCs); however, using a pharmaceutical agent to stimulate hMSC differentiation is not the best choice for engineered tissue transplantation due to potential side-effects. The goal of the present study was to investigate the effects of dynamic compressive loading on differentiation and mineralized matrix production of hMSCs in 3D polyurethane scaffolds, using a loading regimen previously shown to stimulate mineralised matrix production of mature bone cells (MLO-A5). hMSCs were seeded in polyurethane scaffolds and cultured in standard culture media with or without Dex. Cell-seeded scaffolds were compressed at 5% global strain for 2 h on day 9 and then every 5 days in a media-filled sterile chamber. Samples were tested for mRNA expression of alkaline phosphatase (ALP), osteopontin (OPN), collagen type 1 (col 1) and runt-related transcription factor-2 (RUNX-212 h) after the first loading, cell viability by MTS assay and alkaline phosphatase activity at day 12 of culture and cell viability, collagen content by Sirius red and calcium content by alizarin red at day 24 of culture. Neither Dex nor loading had significant effects on cell viability. Collagen content was significantly higher (p<0.01) in the loaded group compared with the non-loaded group in all conditions. There was no difference in ALP activity or the amount of collagen and calcium produced between the non-loaded group supplemented with Dex and the loaded group without Dex. We conclude that dynamic loading has the ability to stimulate osteogenic differentiation of hMSC in the absence of glucocorticoids.

Therapeutic potential of non-adherent BM-derived mesenchymal stem cells in tissue regeneration.

We demonstrated that non-adherent BM cells (NA-BMCs) can be expanded in suspension and give rise to multiple mesenchymal phenotypes including fibroblastic, osteoblastic, chondrocytic and adipocytic as well as glial cell lineages in vitro using the 'pour-off' BMC culture method. Mesenchymal stem cells (MSCs) derived from NA-BMCs (NA-MSCs) from wild-type mice were transplanted into VDR gene knockout (VDR(-/-)) mice that had received a lethal dose of radiation. Results revealed that NA-MSC can be used to rescue lethally irradiated mice and become incorporated into a diverse range of tissues. After lethal dose irradiation, all untransplanted mice died within 2 weeks, whereas those transplanted with NA-MSCs were viable for at least 3 months. Transplantation rescued these mice by reconstructing a hematopoietic system and repairing other damaged tissues. WBC, RBC and platelet counts recovered to normal after 1 month, and VDR gene expression was found in various tissues of viable VDR(-/-) recipients. Adult BM harbors pluripotent NA-MSCs, which can migrate in vivo into multiple body organs. In an appropriate microenvironment, they can adhere, proliferate and differentiate into specialized cells of target tissues and thus function in damaged tissue regeneration and repair.

Tissue specific characteristics of cells isolated from human and rat tendons and ligaments.

BACKGROUND: Tendon and ligament injuries are common and costly in terms of surgery and rehabilitation. This might be improved by using tissue engineered constructs to accelerate the repair process; a method used successfully for skin wound healing and cartilage repair. Progress in this field has however been limited; possibly due to an over-simplistic choice of donor cell. For tissue engineering purposes it is often assumed that all tendon and ligament cells are similar despite their differing roles and biomechanics. To clarify this, we have characterised cells from various tendons and ligaments of human and rat origin in terms of proliferation, response to dexamethasone and cell surface marker expression. METHODS: Cells isolated from tendons by collagenase digestion were plated out in DMEM containing 10% fetal calf serum, penicillin/streptomycin and ultraglutamine. Cell number and collagen accumulation were by determined methylene blue and Sirius red staining respectively. Expression of cell surface markers was established by flow cytometry. RESULTS: In the CFU-f assay, human PT-derived cells produced more and bigger colonies suggesting the presence of more progenitor cells with a higher proliferative capacity. Dexamethasone had no effect on colony number in ACL or PT cells but 10 nM dexamethasone increased colony size in ACL cultures whereas higher concentrations decreased colony size in both ACL and PT cultures. In secondary subcultures, dexamethasone had no significant effect on PT cultures whereas a stimulation was seen at low concentrations in the ACL cultures and an inhibition at higher concentrations. Collagen accumulation was inhibited with increasing doses in both ACL and PT cultures. This differential response was also seen in rat-derived cells with similar differences being seen between Achilles, Patellar and tail tendon cells. Cell surface marker expression was also source dependent; CD90 was expressed at higher levels by PT cells and in both humans and rats whereas D7fib was expressed at lower levels by PT cells in humans. CONCLUSION: These data show that tendon & ligament cells from different sources possess intrinsic differences in terms of their growth, dexamethasone responsiveness and cell surface marker expression. This suggests that for tissue engineering purposes the cell source must be carefully considered to maximise their efficacy.

Age-related changes in human bone marrow-derived mesenchymal stem cells: consequences for cell therapies.

Human mesenchymal stem cells (hMSC) represent a promising cell-based therapy for a number of degenerative conditions. Understanding the effect of aging on hMSCs is crucial for autologous therapy development in older subject whom degenerative diseases typically afflict. Previous investigations into the effects of aging on hMSC have proved contradictory due to the relative narrow age ranges of subjects assessed and the exclusive reliance of in vitro assays. This study seeks to address this controversy by using a wider range of donor ages and by measuring indices of cellular aging as well as hMSC numbers ex vivo and proliferation rates. CFU-f analysis and flow cytometry analysis using a CD45(low)/D7fib(+ve)/LNGF(+ve) gating strategy were employed. In addition a variety of markers of cellular aging, oxidative damage and senescence measured. A reduction in CFU-f and CD45(low)/D7fib(+ve)/LNGF(+ve) cell numbers were noted in adulthood relative to childhood. Indices of aging including oxidative damage, ROS levels and p21 and p53 all increased suggesting a loss of MSC fitness with age. These data suggest that hMSC numbers obtained by marrow aspiration decline with age. Furthermore, there is an age-related decline in overall BM MSC "fitness" which might lead to problems when using autologous aged MSC for cell-based therapies.

Development, optimization and characterization of a full-thickness tissue engineered human oral mucosal model for biological assessment of dental biomaterials.

Restorative dental materials and oral health care products come into direct contact with oral mucosa and can cause adverse reactions. In order to obtain an accurate risk assessment, the in vitro test model must reflect the clinical situation as closely as possible. The aim of this study was to develop and optimize a three-dimensional full-thickness engineered human oral mucosal model, which can be used for biological assessment of dental materials. In this study human oral fibroblasts and keratinocytes were isolated from patients and seeded onto a number of collagen-based and synthetic scaffolds using a variety of cell seeding techniques and grown at the air/liquid interface to construct human oral mucosa equivalents. Suitability of 10 different scaffolds for engineering human oral mucosa was evaluated in terms of biocompatibility, biostability, porosity, and the ability to mimic normal human oral mucosa morphology. Finally an optimized full-thickness engineered human oral mucosa was developed and characterized using transmission electron microscopy and immunostaining. The oral mucosa reconstruct resembled native human oral mucosa and it has the potential to be used as an accurate and reproducible test model in mucotoxicity and biocompatibility evaluation of dental materials.

HPLC analysis of components released from dental composites with different resin compositions using different extraction media.

Components released from dental composite resins are essential factors in the assessment of biocompatibility of these materials. The effect of different extraction media on monomer release from composite resins based on different monomer types was evaluated. Three types of visible light cured composite resins were formulated based on the following monomers: triethylene glycol dimethacrylate (TEGDMA), bisphenol A glycerolate dimethacrylate (BisGMA), and urethane dimethacrylate (UDMA). Seventy-five composite resin discs were fabricated and light cured for 1 min in the absence of oxygen. Extraction media used were: distilled water, saline solution, artificial saliva, serum-free culture medium, and culture medium with 10% fetal calf serum. The analysis of extracts from the composite resins was carried out by High Performance Liquid Chromatography (HPLC). Quantifiable amounts of TEGDMA were released into the aqueous media. However, BisGMA and UDMA were not detectable in any of the extracts from the composite resins. Statistical analysis by one-way ANOVA followed by Tukey's test showed that there was a significant difference in TEGDMA release between culture media and other media (p < 0.05). From the results of this experiment it can be concluded that TEGDMA-based composite resins can release a high quantity of monomer into aqueous environments. The type of extraction medium may have a significant effect on monomer release from composite resins.

Tissue-engineered oral mucosa: a review of the scientific literature.

Tissue-engineered oral mucosal equivalents have been developed for clinical applications and also for in vitro studies of biocompatibility, mucosal irritation, disease, and other basic oral biology phenomena. This paper reviews different tissue-engineering strategies used for the production of human oral mucosal equivalents, their relative advantages and drawbacks, and their applications. Techniques used for skin tissue engineering that may possibly be used for in vitro reconstruction of human oral mucosa are also discussed.

Cannabinoids stimulate fibroblastic colony formation by bone marrow cells indirectly via CB2 receptors.

Recently, the cannabinoid receptors CB(1) and CB(2) were shown to modulate bone formation and resorption in vivo, although little is known of the mechanisms underlying this. The effects of cannabinoids on mesenchymal stem cell (MSC) recruitment in whole bone marrow were investigated using either the fibroblastic colony-forming unit (CFU-f) assay or high-density cultures of whole bone marrow. Levels of the CB(1) and CB(2) receptors were assessed by flow cytometry. Treatment of CFU-f cultures with the endocannabinoid 2-arachidonylglycerol (2-AG) dose-dependently increased fibroblastic and differentiated colony formation along with colony size. The nonspecific agonists CP 55,940 and WIN 55,212 both increased colony numbers, as did the CB(2) agonists BML190 and JWH015. The CB(1)-specific agonist ACEA had no effect, whereas the CB(2) antagonist AM630 blocked the effect of the natural cannabinoid tetrahydrocannabivarin, confirming mediation via the CB(2) receptor. Treatment of primary bone marrow cultures with 2-AG stimulated proliferation and collagen accumulation, whereas treatment of subcultures of MSC had no effect, suggesting that the target cell is not the MSC but an accessory cell present in bone marrow. Subcultures of MSCs were negative for CB(1) and CB(2) receptors as shown by flow cytometry, whereas whole bone marrow contained a small population of cells positive for both receptors. These data suggest that cannabinoids may stimulate the recruitment of MSCs from the bone marrow indirectly via an accessory cell and mediated via the CB(2) receptor. This recruitment may be one mechanism responsible for the increased bone formation seen after cannabinoid treatment in vivo.

Effects of phenol red on CFU-f differentiation and formation.

Osteoblastic induction is commonly studied using the colony-forming unit-fibroblastic (CFU-f) assay, in which bone marrow stromal cells (BMC) are grown in a tissue culture environment permissive for osteoblastic differentiation (DMEM containing dexamethasone, ascorbic acid and beta-glycerophosphate). These cells form colonies, which express alkaline phosphatase, and form a collagenous matrix that becomes calcified. However, these same cells originate in the bone marrow where under normal circumstances they do not proliferate or differentiate despite being subjected to many of the same growth factors and hormones present within the tissue culture environment. We show here that phenol red, present within tissue culture medium as a pH indicator, may itself be a factor that permits osteoblastic recruitment. BMC cultured in the presence of the bone anabolic agents PGE2, PGA2, or bFGF, but in the absence of phenol red, failed to respond to these agents in terms of total or osteoblastic colony number. This effect was dose dependent, with low (2.5 mg/l) and high (15-20 mg/l) doses of phenol red being nonpermissive for the stimulatory effects of PGE2 whereas doses of 5-10 mg/l were permissive. Furthermore, the effects observed in the absence of phenol red could not be abrogated by the addition of 17beta-estradiol indicating that these effects cannot be attributed to estrogenic impurities within the phenol red preparation. This indicates that phenol red itself can affect the differentiation of BMC by a mechanism not previously described.

Effects of risedronate, alendronate, and etidronate on the viability and activity of rat bone marrow stromal cells in vitro.

The effects of risedronate, alendronate, and etidronate were assessed in calcifying fibroblastic colony-forming unit (CFU-f) cultures of rat bone marrow cells in vitro. Biphasic effects on the formation of bone-like colonies were observed. Treatment with high concentrations (10(-5)-10(-4)M) of alendronate and risedronate caused a total inhibition of colony formation whereas etidronate had relatively little effect. It was also found that intermediate concentrations (10(-6)M) of alendronate and risedronate decreased the formation of colonies displaying osteoblastic characteristics such as alkaline phosphatase expression, collagen accumulation, and calcification. At lower concentrations (10(-9)-10(-7)M), risedronate and alendronate increased the formation of fibroblastic colonies, suggesting a mild anabolic effect, however, the formation of colonies with osteoblastic properties was not affected. Treating the cells with a combination of bisphosphonate and 1 mM geranylgeraniol could to some extent abrogate the cytotoxic effects of alendronate or risedronate, suggesting the involvement of the mevalonate pathway. The colony-stimulating activity of these bisphosphonates was, however, unaffected.

Novel therapeutic targets in osteoporosis.

Osteoporosis is a common condition in which significant bone loss occurs resulting in an increased risk of sustaining fractures. Several licensed therapies are available to treat this condition, which suffer from several disadvantages including limited efficacy, high cost and poor long-term patient adherence as a consequence of significant side effects and inconvenient methods of administration. A wide range of therapeutic targets have been developed to provide a basis for developing newer therapies which overcome these limitations. These can be subdivided into those that are primarily directed towards inhibiting osteoclast-dependent bone resorption and those that stimulate osteoblastic bone formation. Targets can be grouped as follows: systemic factors such as steroid and peptide hormones; local factors produced in bone involved in osteoblast and osteoclastic regulation; and cellular targets such as cell membrane receptors and attachment proteins, cellular enzymes and nuclear transcription factors. To date, only a small proportion of these targets have yielded novel compounds to have entered clinical trials. However, it is anticipated that these will provide the basis for significant numbers of new therapies for osteoporosis in the foreseeable future.

Stimulation of CFU-f formation by prostaglandin E2 is mediated in part by its degradation product, prostaglandin A2.

Prostaglandins (PG) of the E series are known to rapidly undergo non-enzymatic dehydration in culture medium containing serum albumin to produce the cyclopentenone PGs of the A series. We investigated the actions of PGA1 and A2 in the in vitro calcifying fibroblastic-colony forming unit assay which can partially mimic the in vivo anabolic effects of PGE2. It was found that PGA1 and A2 both stimulated colony formation in a dose-dependent manner with a maximum at 10(-6) M and to a similar degree to PGE2. In contrast to PGE2, PGA1 and PGA2 both caused an inhibition of cAMP accumulation. Furthermore, the addition of protein kinase A inhibitors, H8 and H89, had no significant effect on the stimulation of colony number by PGE2. These data suggest that (a) the bone anabolic effects of PGE1 and E2 are, in part at least, mediated by their dehydration products PGA1 and A2 and (b) that they are mediated via pathways not necessarily involving the cAMP/protein kinase A cascade.

Mild heat shock induces proliferation, alkaline phosphatase activity, and mineralization in human bone marrow stromal cells and Mg-63 cells in vitro.

Bone formation has been shown to be stimulated by local diathermy in vivo; however, the mechanisms involved in this heat-induced osteogenesis are unclear. In this study, we investigated the direct effect of temperature on human bone marrow-derived stromal cells (BMSCs) and the human osteoblast-like, osteosarcoma-derived MG-63 cells in culture conditions. Both cell types were shown to tolerate the transient exposure to mild heat shock conditions (1 h at 39-41 degrees C), and long-term (96 h) exposure at 39 degrees C stimulated DNA synthesis in BMSC but caused growth arrest in MG-63 cells. Furthermore, 1-h exposure to higher temperatures (42.5-45 degrees C) or continuous 96-h exposure to 40 degrees C or 41 degrees C inhibited the proliferation of both BMSCs and MG63 cells. The level of alkaline phosphatase (ALP) in these cells linearly correlated with the increase in temperature, and the ALP expression, either at the basal level or in response to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], was enhanced after a single 1-h exposure to 42.5 degrees C. In addition, continuous incubation at 39 degrees C or repeated transient exposure to 39/41 degrees C greatly enhanced the ability of BMSCs to form mineralizing nodules. The heat shock protein HSP70, which was expressed constitutively by BMSCs, was found to be up-regulated by hyperthermia (39 degrees C) and down-regulated at 33 degrees C. The expression of HSP70 could be induced in MG-63 cells by both low- and high-temperature conditions. These data suggest that treatment with a mild heat shock induces the proliferation and differentiation of osteoprogenitor cells, and the direct effects of temperature on bone-forming cells might be one of the mechanisms involved in heat-induced bone formation in vivo.

Human myeloma cells promote the recruitment of osteoblast precursors: mediation by interleukin-6 and soluble interleukin-6 receptor.

Multiple myeloma is associated with the development of osteolytic bone disease characterized by a disruption to normal bone resorption and bone formation. Although studies have shown that myeloma cells produce factors that promote bone resorption little data are available examining the mechanism of decreased bone formation or the factors that mediate this effect. In the present study we describe a novel in vitro coculture system in which to investigate the effect of myeloma cells on osteoblast recruitment and differentiation. Under appropriate conditions mesenchymal stem cells were shown to differentiate into colonies of cells, a proportion of which show characteristics of osteoblasts, in that they express alkaline phosphatase activity and stain positively for collagen and calcium. The addition of the human myeloma cells JJN-3, RPMI-8226, or NCI-H929 to these cultures stimulated a significant increase in the total number of colonies (p < 0.005) and the proportion of osteoblastic colonies (p < 0.005). Media conditioned by these cells also were able to promote the formation of both total and osteoblastic colonies (p < 0.005). The addition of an antibody against the interleukin-6 receptor (IL-6R) blocked myeloma cell and myeloma cell-conditioned media induced osteoblast recruitment (p < 0.01). Furthermore, media conditioned by myeloma cells incubated with phorbol ester, which promotes IL-6R shedding, or a metalloproteinase inhibitor, which inhibits IL-6R shedding, were able to stimulate (p < 0.005) and inhibit osteoblast recruitment (p < 0.005), respectively. In addition, soluble IL-6R (sIL-6R) and IL-6 together, but not alone, were able to promote osteoblastic colony formation (p < 0.01). Taken together these data show that myeloma cells promote osteoblast recruitment by release of sIL-6R from myeloma cells.

Centrifugal isolation of bone marrow from bone: an improved method for the recovery and quantitation of bone marrow osteoprogenitor cells from rat tibiae and femurae.

SUMMARY. The high variation often observed in the ex vivo fibroblastic-colony forming unit (CFU-f) assay is likely to be due to both biological and experimental variation. To determine whether we could improve experimental methods we developed an alternative method of bone marrow cell (BMC) isolation employing a centrifugation step. The osteogenic capacity of centrifugally isolated BMC was compared to that of BMC that were isolated using the standard "flushing" technique using the CFU-f assay. The centrifugation method was found to be both quick and simple to perform and allowed simultaneous preparation of all samples. Centrifugally isolated BMC gave rise to approximately 100% more cfu-ap and cfu-f in cultures from both tibiae and femurae. The proportion of alkaline phosphatase positive colonies remained the same and colony morphologies were similar for both isolation methods. Histological comparison of the flushed and spun bones showed that after the flushing procedure many cells remained in the marrow cavity especially in the trabecular area. In contrast, centrifugation completely emptied the marrow space of all cells except bone lining cells and osteoblasts. Thus the osteogenic capacity of the bone marrow can be expressed as the number of CFU-f per bone instead of the frequency as is the norm. Using these methods to isolate BMC for ex vivo investigations should lead to a reduction in CFU-f number variation due to the isolation method. http://link.springer-ny. com/link/service/journals/00223/bibs/65n5p411.html

A cost-effective method for the automatic quantitative analysis of fibroblastic colony-forming units.

A great deal of the work characterizing stromal cell precursors in the bone marrow has been performed using the fibroblastic colony-forming unit (CFU-f) assay. However, the assay is limited in its usefulness by the necessity for manual colony counting which means that assay quantitation is highly subjective, time consuming, and much information regarding the colony size is lost. To rectify this, we have developed a computer-automated method for the analysis of CFU-f. Bone marrow cells were cultured at low density and treated with either prostaglandin E(2) (PGE(2)), basic fibroblast growth factor (bFGF), or dexamethasone, and colony formation was assessed by staining with methylene blue. After staining, the dishes were photographed over a light box using a digital camera and the image was then analyzed using Bioimage "Intelligent Quantifier" image analysis software which automatically locates and quantifies each individual colony. The data can then be imported to a spreadsheet program and processed. We have shown that this system can accurately identify, assign coordinates, and quantitate each individual colony. Colony numbers obtained with this method and manually counting showed a linear relationship with a correlation coefficient of 0.99. In addition, using the colony intensity and surface area data, the colony size can be calculated. With this methodology, we have shown that dexamethasone, PGE(2), and bFGF can all modulate total cell numbers in bone marrow stromal cells (BMSC) cultures but modulating both colony number and colony size.

Regulation of osteogenic differentiation of human bone marrow stromal cells: interaction between transforming growth factor-beta and 1,25(OH)(2) vitamin D(3) In vitro.

Bone marrow stromal cells are believed to play a major role in bone formation as a major source of osteoprogenitor cells, however, very little is known about how the osteogenic differentiation of these cells is regulated by systemic hormones and local growth factors. We examined the effects of TGF-beta and its interaction with 1, 25(OH)(2) Vitamin D(3) [1,25(OH)(2)D(3)] on the differentiation and proliferation of human bone marrow stromal cells (hBMSC) in secondary cultures. Alkaline phosphatase (ALP) activity was inhibited by TGF-beta (0.1-10 ng/ml) and increased by 1, 25(OH)(2)D(3) (50 nM), however, co-treatment of TGF-beta and 1, 25(OH)(2)D(3) synergistically enhanced ALP activity with maximal stimulation occurring at about 8 days after treatment. This synergistic effect was independent of proliferation because, in contrast to TGF-beta alone, combined treatment with TGF-beta and 1, 25(OH)(2)D(3) had no effect on hBMSC proliferation. As no synergistic effect was seen with combinations of 1,25(OH)(2)D(3) and other osteotrophic growth factors, including BMP-2, IGF-I, and basic fibroblast growth factor (bFGF), it would seem likely that the synergistic interaction is specific for TGF-beta. The increased ALP activity was due to an enhancement of 1,25(OH)(2)D(3)-induced ALP activity by TGF-beta, rather than vice versa. In contrast, TGF-beta inhibited 1,25(OH)(2)D(3)-induced osteocalcin production. Taken together, these results indicate that TGF-beta and 1,25(OH)(2)D(3) act synergistically to stimulate the recruitment of BMSC to the osteoblast lineage. This interaction may play an important role in bone remodeling.

Basic fibroblast growth factor in the presence of dexamethasone stimulates colony formation, expansion, and osteoblastic differentiation by rat bone marrow stromal cells.

Basic fibroblast growth factor (bFGF) is known to stimulate endosteal bone formation in vivo by a mechanism possibly mediated via osteoblast precursor cells present in the bone marrow. In high density cultures of primary bone marrow cells, and in the presence of glucocorticoids, bFGF stimulates the formation of a bone-like matrix; however, due to the dense nature of these cultures, the exact mechanism of action is unclear. In an adaptation of the fibroblastic colony formation unit assay, in which the bone marrow cells are grown in the presence of dexamethasone, beta-glycerophosphate, and ascorbate, mineralized colonies are formed which stem from single mesenchymal precursor cells and grow in isolation from each other. Using this system we have been able to investigate the mechanism by which bFGF stimulates the formation of bone like tissue in vitro. We have shown that bFGF increases the formation of a calcified collagenous matrix in vitro by (1) increasing the total number of fibroblastic colonies formed, (2) increasing the proportion of differentiated colonies that synthesize collagen and calcify, and (3) stimulating the proliferation and collagen accumulation of the individual colonies. A maximal increase in total and differentiated colony numbers was seen after only 5 days exposure to bFGF, however, continued exposure to bFGF continued to increase the size and collagen content of the individual colonies. Bearing in mind the endosteal location of newly formed bone seen after treatment with bFGF, these processes may well play an active role in this effect.