Mesenchymal stem cells: building blocks for molecular medicine in the 21st century.

Mesenchymal stem sells (MSCs) are present in a variety of tissues during human development, and in adults they are prevalent in bone marrow. From that readily available source, MSCs can be isolated, expanded in culture, and stimulated to differentiate into bone, cartilage, muscle, marrow stroma, tendon, fat and a variety of other connective tissues. Because large numbers of MSCs can be generated in culture, tissue-engineered constructs principally composed of these cells could be re-introduced into the in vivo setting. This approach is now being explored to regenerate tissues that the body cannot naturally repair or regenerate when challenged. Moreover, MSCs can be transduced with retroviral and other vectors and are, thus, potential candidates to deliver somatic gene therapies for local or systemic pathologies. Untapped applications include both diagnostic and prognostic uses of MSCs and their descendents in healthcare management. Finally, by understanding the complex, multistep and multifactorial differentiation pathway from MSC to functional tissues, it might be possible to manipulate MSCs directly in vivo to cue the formation of elaborate, composite tissues in situ.

Incipient analysis of mesenchymal stem-cell-derived osteogenesis.

Tissue regeneration strategies invoke cell-based therapies for effective tissue formation. Current assessment of mesenchymal stem cell (MSC) directed bone regeneration during in vivo assays is dependent on histologic determination of bone formation. It was the aim of this study to determine the relationship between bone sialoprotein (BSP) expression and osteocalcin expression with subsequent osteogenesis occurring in MSC-based implants. RT-PCR assessment of human actin, collagen type I, BSP, and osteocalcin indicated that undifferentiated cells did not express BSP or osteocalcin. Three weeks following implantation, human BSP could be identified in RNAs isolated from the retrieved implants. For every implant from which human BSP cDNA was amplified, parallel implants harvested at 6 weeks demonstrated bone formation at the histologic level. This study confirms that, in the context of the severe combined immunodeficiency disease (SCID) mouse model, culture-expanded, cryopreserved human MSCs have osteogenic potential and demonstrates that implanted cell gene expression can reveal the early onset of bone formation.

Adult human mesenchymal stem cell differentiation to the osteogenic or adipogenic lineage is regulated by mitogen-activated protein kinase.

Adult human mesenchymal stem cells are primary, multipotent cells capable of differentiating to osteocytic, chondrocytic, and adipocytic lineages when stimulated under appropriate conditions. To characterize the molecular mechanisms that regulate osteogenic differentiation, we examined the contribution of mitogen-activated protein kinase family members, ERK, JNK, and p38. Treatment of these stem cells with osteogenic supplements resulted in a sustained phase of ERK activation from day 7 to day 11 that coincided with differentiation, before decreasing to basal levels. Activation of JNK occurred much later (day 13 to day 17) in the osteogenic differentiation process. This JNK activation was associated with extracellular matrix synthesis and increased calcium deposition, the two hallmarks of bone formation. Inhibition of ERK activation by PD98059, a specific inhibitor of the ERK signaling pathway, blocked the osteogenic differentiation in a dose-dependent manner, as did transfection with a dominant negative form of MAP kinase kinase (MEK-1). Significantly, the blockage of osteogenic differentiation resulted in the adipogenic differentiation of the stem cells and the expression of adipose-specific mRNAs peroxisome proliferator-activated receptor gamma2, aP2, and lipoprotein lipase. These observations provide a potential mechanism involving MAP kinase activation in osteogenic differentiation of adult stem cells and suggest that commitment of hMSCs into osteogenic or adipogenic lineages is governed by activation or inhibition of ERK, respectively.

Role of the latent transforming growth factor beta binding protein 1 in fibrillin-containing microfibrils in bone cells in vitro and in vivo.

Latent transforming growth factor beta-binding proteins (LTBPs) are extracellular matrix (ECM) proteins that bind latent transforming growth factor beta (TGF-beta) and influence its availability in bone and other connective tissues. LTBPs have homology with fibrillins and may have related functions as microfibrillar proteins. However, at present little is known about their structural arrangement in the ECM. By using antibodies against purified LTBP1, against a short peptide in LTBP1, and against epitope-tagged LTBP1 constructs, we have shown colocalization of LTBP1 and fibrillin 1 in microfibrillar structures in the ECM of cultured primary osteoblasts. Immunoelectron microscopy confirmed localization of LTBP1 to 10- to 12-nm microfibrils and suggested an ordered aggregation of LTBP1 into these structures. Early colocalization of LTBP1 with fibronectin suggested a role for fibronectin in the initial assembly of LTBP1 into the matrix; however, in more differentiated osteoblast cultures, LTBP1 and fibronectin 1 were found in distinct fibrillar networks. Overexpression of LTBP1 deletion constructs in osteoblast-like cells showed that N-terminal amino acids 67-467 were sufficient for incorporation into fibrillin-containing microfibrils and suggested that LTBP1 can be produced by cells distant from the site of fibril formation. In embryonic long bones in vivo, LTBP1 and fibrillin 1 colocalized at the surface of newly forming osteoid and bone. However, LTBP1-positive fibrils, which did not contain fibrillin 1, were present in cartilage matrix. These studies show that in addition to regulating TGF beta 1, LTBP1 may function as a structural component of connective tissue microfibrils. LTBP1 may therefore be a candidate gene for Marfan-related connective tissue disorders in which linkage to fibrillins has been excluded.

Human marrow-derived mesenchymal stem cells (MSCs) express hematopoietic cytokines and support long-term hematopoiesis when differentiated toward stromal and osteogenic lineages.

Human mesenchymal stem cells (MSCs), bone marrow-derived pluripotent adherent cells of mesenchymal origin can differentiate along the osteogenic, chondrogenic, adipogenic, and tendonogenic lineages. In this report we characterize cytokine and growth factor gene expression by MSCs and investigate the modulation of cytokine expression that occurs during osteogenic and stromal differentiation. MSCs constitutively expressed mRNA for interleukin (IL)-6, IL-11, leukemia inhibitory factor (LIF), macrophage colony-stimulating factor (M-CSF), and stem cell factor (SCF). MSCs treated with IL-1alpha upregulated mRNA levels of IL-6, IL-11, and LIF, and began to express detectable levels of granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF). mRNA levels of M-CSF and SCF did not change. MSCs cultured in osteogenic medium differentiated along the osteogenic lineage and downregulated mRNA levels of IL-6, IL-11 and LIF whereas, M-CSF and SCF expression were unchanged and G-CSF and GM-CSF remained undetectable. IL-3 was not detected in MSC culture under any conditions. MSCs precultured in control medium, IL-1alpha, or osteogenic medium maintained similar capacity to support long-term culture initiating cell (LT-CIC). Thus, primary and osteogenic differentiated MSCs produce important hematopoietic cytokines and support hematopoiesis in long-term cultures, suggesting that these cells may provide an excellent ex vivo environment for hematopoiesis during progenitor cell expansion and may be important for in vivo cell therapy.

Tissue engineering of bone. Cell based strategies.

Skeletal tissue regeneration requires the interaction of three basic biologic elements: cells, growth and differentiation factors, and extracellular matrix scaffolds. Therapeutic approaches for tissue engineered repair of bone defects have attempted to mimic the natural process of bone repair by delivering a source of cells capable of differentiating into osteoblasts, inductive growth and differentiation factors, or bioresorbable scaffolding matrices to support cellular attachment, migration, and proliferation. Sophisticated designs even have tried to combine two or more of these elements. The development of cell based approaches has advanced dramatically in recent years as an understanding of musculoskeletal cell biology improves. Cell based approaches do not depend on the presence of local osteoprogenitors for the synthesis of new bone and, as a result, they particularly are attractive for patients who have a diminished pool of these progenitors, or in whom the host tissue bed has been compromised. This review highlights the development of cell based approaches for the tissue engineering of bone, and offers perspectives on the optimal elements for success. Although logistical and regulatory issues remain to be solved, cell based therapies for the repair of clinically significant bone defects rapidly are approaching clinical feasibility.

The effect of implants loaded with autologous mesenchymal stem cells on the healing of canine segmental bone defects.

Bone marrow has been shown to contain a population of rare mesenchymal stem cells that are capable of forming bone, cartilage, and other connective tissues. We examined the effect of cultured autologous mesenchymal stem cells on the healing of critical-sized (twenty-one-millimeter-long) segmental defects in the femora of adult female dogs. Autologous mesenchymal stem cells were isolated from bone marrow, grown in culture, and loaded onto porous ceramic cylinders consisting of hydroxyapatite (65 per cent) and beta-tricalcium phosphate ceramic (35 per cent). The animals were randomly assigned to one of three groups. In Group A (six dogs), a porous ceramic cylinder that had been loaded with autologous mesenchymal stem cells was implanted in the defect. In Group B (six dogs), a ceramic cylinder that had not been loaded with cells was placed in the defect. In Group C (three dogs), the defect was left untreated (no ceramic cylinder was implanted). Radiographs were made immediately after the operation and at four-week intervals. At sixteen weeks, the animals were killed, the involved femora were removed, and undecalcified histological sections from the defects and adjacent bone were prepared. Histological and histomorphometric studies were carried out to examine the healing of the defects and the formation of bone in and around the ceramic implants. Atrophic non-union occurred in all of the femora that had untreated defects, and only a small amount of trabecular bone formed at the cut ends of the cortex of the host bone in this group. In contrast, radiographic union was established rapidly at the interface between the host bone and the implants that had been loaded with mesenchymal stem cells. Numerous fractures, which became more pronounced with time, developed in the implants that had not been loaded with cells. Histological and morphometric analyses demonstrated that both woven and lamellar bone had filled the pores of the implants that had been loaded with mesenchymal stem cells; the amount of bone was significantly greater (p < 0.05) than that found in the pores of the implants that had not been loaded with cells. In addition, a large collar of bone (mean maximum thickness, 3.14 millimeters) formed around the implants that had been loaded with cells; this collar became integrated and contiguous with callus that formed in the region of the periosteum of the host bone. The collar of bone remodeled during the sixteen-week period of study, resulting in a size and shape that were comparable with those of the segment of bone that had been resected. Callus did not develop around the cortex of the host bone or around the defect in any of the specimens in the other two groups.

Bone regeneration by implantation of purified, culture-expanded human mesenchymal stem cells.

Bone marrow contains a population of rare progenitor cells capable of differentiating into bone, cartilage, tendon, and other connective tissues. These cells, referred to as mesenchymal stem cells, can be purified and culture-expanded from animals and humans and have been shown to regenerate functional tissue when delivered to the site of musculoskeletal defects in experimental animals. To test the ability of purified human mesenchymal stem cells to heal a clinically significant bone defect, mesenchymal stem cells isolated from normal human bone marrow were culture-expanded, loaded onto a ceramic carrier, and implanted into critical-sized segmental defects in the femurs of adult athymic rats. For comparison, cell-free ceramics were implanted in the contralateral limb. The animals were euthanized at 4, 8, or 12 weeks, and healing bone defects were compared by high-resolution radiography, immunohistochemistry, quantitative histomorphometry, and biomechanical testing. In mesenchymal stem cell-loaded samples, radiographic and histologic evidence of new bone was apparent by 8 weeks and histomorphometry demonstrated increasing bone formation through 12 weeks. Biomechanical evaluation confirmed that femurs implanted with mesenchymal stem cell-loaded ceramics were significantly stronger than those that received cell-free ceramics. These studies demonstrate that human mesenchymal stem cells can regenerate bone in a clinically significant osseous defect and may therefore provide an alternative to autogenous bone grafts.

Mesenchymal stem cell surface antigen SB-10 corresponds to activated leukocyte cell adhesion molecule and is involved in osteogenic differentiation.

Bone marrow contains a rare population of mesenchymal stem cells (MSCs) capable of giving rise to multiple mesodermal tissues including bone, cartilage, tendon, muscle, and fat. The cell surface antigen recognized by monoclonal antibody SB-10 is expressed on human MSCs but is lost during their developmental progression into differentiated phenotypes. Here we report on the immunopurification of the SB-10 antigen and its identification as activated leukocyte-cell adhesion molecule (ALCAM). Mass spectrometry establishes that the molecular mass of ALCAM is 80,303 +/- 193 Da and that it possesses 17,763 +/- 237 Da of N-linked oligosaccharide substituents. Molecular cloning of a full-length cDNA from a MSC expression library demonstrates nucleotide sequence identity with ALCAM. We also identified ALCAM homologs in rat, rabbit, and canine MSCs, each of which is over 90% identical to human ALCAM in their peptide sequence. The addition of antibody SB-10 Fab fragments to human MSCs undergoing osteogenic differentiation in vitro accelerated the process, thereby implicating a role for ALCAM during bone morphogenesis and adding ALCAM to the group of cell adhesion molecules involved in osteogenesis. Together, these results provide evidence that ALCAM plays a critical role in the differentiation of mesenchymal tissues in multiple species across the phylogenetic tree.

Mesenchymal stem cells in osteobiology and applied bone regeneration.

Bone marrow contains a population of rare progenitor cells capable of differentiating into bone, cartilage, muscle, tendon, and other connective tissues. These cells, referred to as MSCs, can be purified and culture expanded from animals and humans. This review summarizes recent experimentation focused on characterizing the cellular aspects of osteogenic differentiation, and exploration of the potential for using autologous stem cell therapy to augment bone repair and regeneration. The authors have completed an array of preclinical studies showing the feasibility and efficacy of MSC based implants to heal large osseous defects. After confirming that syngeneic rat MSCs could heal a critical size segmental defect in the femur, it was established that human MSCs form bone of considerable mechanical integrity when implanted in an osseous defect in an immunocompromised animal. Furthermore, bone repair studies in dogs verify that the technology is transferable to large animals, and that the application of this technology to patients at geographically remote sites is feasible. These studies suggest that by combining MSCs with an appropriate delivery vehicle, it may be possible to offer patients new therapeutic options.

Monoclonal antibodies reactive with human osteogenic cell surface antigens.

Monoclonal antibodies (McAbs) against the surface of osteoblastic cells have been used to characterize the osteogenic lineage. In view of the paucity of probes against the surface of normal human osteogenic cells, we sought to generate McAbs which could be used for both in vivo and in vitro studies. We raised a series of McAbs against early osteoblastic cell surface antigens by immunizing mice with human mesenchymal stem cells (MSCs) that had been directed into the osteogenic lineage in vitro. After screening against the surface of osteogenic cells at various stages of differentiation in vitro, as well as evaluating in situ reactivity with human fetal limbs, we isolated three hybridoma cell lines referred to as SB-10, SB-20, and SB-21. Immunocytochemical analyses during osteogenic differentiation demonstrate that SB-10 reacts with MSCs and osteoprogenitors, but no longer reacts with cells once alkaline phosphatase (APase) is expressed. Flow cytometry documents that SB-10 is expressed on the surface of all purified, culture-expanded human MSCs, thus providing further evidence that these cells are a homogeneous population. By contrast, SB-20 and SB-21 do not react with the progenitor cells in situ, but bind to a subset of the APase-positive osteoblasts. None of these antibodies stain terminally differentiated osteocytes in sections of developing bone. Furthermore, these McAbs were not observed to react in samples from chick, rat, rabbit, canine, or bovine bone, although selected extraskeletal human tissues were immunostained. In all cell and tissue specimens examined, SB-20 immunostaining is identical to that observed with SB-21. We have used these McAbs to refine our understanding of the discrete cellular transitions that constitute the osteogenic cell lineage. We suggest a refined model for understanding osteoblast differentiation that is based on the proposition that the sequential acquisition and loss of specific cell surface molecules can be used to define positions of individual cells within the osteogenic cell lineage.

Culture expanded canine mesenchymal stem cells possess osteochondrogenic potential in vivo and in vitro.

Mesenchymal Stem Cells (MSCs) possessing the capacity to differentiate into various cell types such as osteoblasts, chondrocytes, myoblasts, and adipocytes have been previously isolated from the marrow and periosteum of human, murine, lapine, and avian species. This study documents the existence of similar multipotential stem cells in canine marrow. The cells were isolated from marrow aspirates using a modification of techniques previously established for human MSCs (hMSCs), and found to possess similar growth and morphological characteristics, as well as osteochondrogenic potential in vivo and in vitro. On the basis of these results, the multipotential cells that were isolated and culture expanded are considered to be canine MSCs (cMSCs). The occurrence of cMSCs in the marrow was determined to be one per 2.5 x 10(4) nucleated cells. After enrichment of the cMSCs by centrifugation on a Percoll cushion, the cells were cultivated in selected lots of serum. Like the hMSCs, cMSCs grew as colonies in primary culture and on replating, grew as a monolayer culture with very uniform spindle morphology. The population doubling time for these cMSCs was approximately 2 days. The morphology and the growth kinetics of the cMSCs were retained following repeated passaging. The osteogenic phenotype could be induced in the cMSC cultures by the addition of a synthetic glucocorticoid, dexamethasone. In these osteogenic cultures, alkaline phosphatase activity was elevated up to 10-fold, and mineralized matrix production was evident. When cMSCs were loaded onto porous ceramics and implanted in autologous canine or athymic murine hosts, copious amounts of bone and cartilage were formed in the pores of the implants. The MSC-mediated osteogenesis obtained by the implantation of the various MSC-loaded matrix combinations is the first evidence of osteogenesis in a canine model by implantation of culture expanded autologous stem cells. The identification and isolation of cMSCs now makes it feasible to pursue preclinical models of bone and cartilage regeneration in canine hosts.

Growth kinetics, self-renewal, and the osteogenic potential of purified human mesenchymal stem cells during extensive subcultivation and following cryopreservation.

Recent studies have demonstrated the existence of a subset of cells in human bone marrow capable of differentiating along multiple mesenchymal lineages. Not only do these mesenchymal stem cells (MSCs) possess multilineage developmental potential, but they may be cultured ex vivo for many passages without overt expression of a differentiated phenotype. The goals of the current study were to determine the growth kinetics, self-renewing capacity and the osteogenic potential of purified MSCs during extensive subcultivation and following cryopreservation. Primary cultures of MSCs were established from normal iliac crest bone marrow aspirates, an aliquot was cryopreserved and thawed, and then both frozen and unfrozen populations were subcultivated in parallel for as many as 15 passages. Cells derived from each passage were assayed for their kinetics of growth and their osteogenic potential in response to an osteoinductive medium containing dexamethasone. Spindle-shaped human MSCs in primary culture exhibit a lag phase of growth, followed by a log phase, finally resulting in a growth plateau state. Passaged cultures proceed through the same stages, however, the rate of growth in log phase and the final number of cells after a fixed period in culture diminishes as a function of continued passaging. The average number of population doublings for marrow-derived adult human MSCs was determined to be 38 +/- 4, at which time the cells finally became very broad and flattened before degenerating. The osteogenic potential of cells was conserved throughout every passage as evidenced by the significant increase in APase activity and formation of mineralized nodular aggregates. Furthermore, the process of cryopreserving and thawing the cells had no effect on either their growth or osteogenic differentiation. Importantly, these studies demonstrate that replicative senescence of MSCs is not a state of terminal differentiation since these cells remain capable of progressing through the osteogenic lineage. The use of population doubling potential as a measure of biological age suggests that MSCs are intermediately between embryonic and adult tissues, and as such, may provide an in situ source for mesenchymal progenitor cells throughout an adult's lifetime.

Osteogenic differentiation of purified, culture-expanded human mesenchymal stem cells in vitro.

Human bone marrow contains a population of cells capable of differentiating along multiple mesenchymal cell lineages. Recently, techniques for the purification and culture-expansion of these human marrow-derived Mesenchymal Stem Cells (MSCs) have been developed. The goals of the current study were to establish a reproducible system for the in vitro osteogenic differentiation of human MSCs, and to characterize the effect of changes in the microenvironment upon the process. MSCs derived from 2nd or 3rd passage were cultured for 16 days in various base media containing 1 to 1000 nM dexamethasone (Dex), 0.01 to 4 mM L-ascorbic acid-2-phosphate (AsAP) or 0.25 mM ascorbic acid, and 1 to 10 mM beta-glycerophosphate (beta GP). Optimal osteogenic differentiation, as determined by osteoblastic morphology, expression of alkaline phosphatase (APase), reactivity with anti-osteogenic cell surface monoclonal antibodies, modulation of osteocalcin mRNA production, and the formation of a mineralized extracellular matrix containing hydroxyapatite was achieved with DMEM base medium plus 100 nM Dex, 0.05 mM AsAP, and 10 mM beta GP. The formation of a continuously interconnected network of APase-positive cells and mineralized matrix supports the characterization of this progenitor population as homogeneous. While higher initial seeding densities did not affect cell number of APase activity, significantly more mineral was deposited in these cultures, suggesting that events which occur early in the differentiation process are linked to end-stage phenotypic expression. Furthermore, cultures allowed to concentrate their soluble products in the media produced more mineralized matrix, thereby implying a role for autocrine or paracrine factors synthesized by human MSCs undergoing osteoblastic lineage progression. This culture system is responsive to subtle manipulations including the basal nutrient medium, dose of physiologic supplements, cell seeding density, and volume of tissue culture medium. Cultured human MSCs provide a useful model for evaluating the multiple factors responsible for the step-wise progression of cells from undifferentiated precursors to secretory osteoblasts, and eventually terminally differentiated osteocytes.

Variable hormone responsiveness of osteoblast populations isolated at different stages of embryogenesis and its relationship to the osteogenic lineage.

A variable response to 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] hormone treatment was observed for osteoblast cell populations isolated from 12- and 17-day-old embryonic chick calvariae. The younger embryonic cell population showed 2- and 5-fold inductions of osteocalcin and osteopontin gene expression, respectively, and a 25% inhibition of collagen gene expression when treated with 1,25-(OH)2D3. In contrast, these same genes all displayed approximately 80% inhibition of their expression when the older embryonic cell populations were treated with hormone. The hormone response was related to the appearance of the vitamin D3 receptor (VDR) and the developmental state of teh two cell populations by assessing the numbers of cells that were immunologically labeled for two osteoblast lineage, stage-specific surface makers (alkaline phosphatase and SB-5, an osteocyte marker) and the VDR. Using the sequence of marker presentation, with VDR appearing first, followed by alkaline phosphatase and then SB-5, models were tested using logistic regression analysis to validate this order of marker presentation and establish that the two embryonic ages of the cell populations represent discrete stages of their lineage. This analysis indicated that 1,25-(OH)2D3 treatment progressed the 12-day-old embryo cell populations along their lineage and that the hormone promoted the appearance of its own receptor (P < 0.001) However, the appearance of the VDR does not appear to be a determinant in the variable responses of the different embryonic aged cell populations to the hormone. These data quantitatively establish the unique nature of osteoblast cell populations within their lineage progression for cells isolated from embryos of different ages, such that cell populations isolated from younger embryos are comprised of primarily presumptive or immature osteoblasts, whereas cells isolated from older embryos are comprised of mature osteoblasts. These data also demonstrate that the genomic effects of 1,25-(OH)2D3 are dependent on the developmental stage of the osteoblast lineage, and the stimulatory actions of the hormone are targeted to immature osteoblasts, whereas the effect of the hormone on mature osteoblasts is inhibitory.

Mesenchymal stem cells in bone development, bone repair, and skeletal regeneration therapy.

Bone formation in the embryo, and during adult fracture repair and remodeling, involves the progeny of a small number of cells called mesenchymal stem cells (MSCs). These cells continuously replicate themselves, while a portion become committed to mesenchymal cell lineages such as bone, cartilage, tendon, ligament, and muscle. The differentiation of these cells, within each lineage, is a complex multistep pathway involving discrete cellular transitions much like that which occurs during hematopoiesis. Progression from one stage to the next depends on the presence of specific bioactive factors, nutrients, and other environmental cues whose exquisitely controlled contributions orchestrate the entire differentiation phenomenon. An understanding of the cellular and molecular events of osteogenic differentiation of MSCs provides the foundation for the emergence of a new therapeutic technology for cell therapy. The isolation and in vitro mitotic expansion of autologous human MSCs will support the development of novel protocols for the treatment of many clinically challenging conditions. For example, local bone defects can be repaired through site-directed delivery of MSCs in an appropriate carrier vehicle. Generalized conditions, such as osteoporosis, may be treatable by systemic administration of culture-expanded autologous MSCs or through biopharmaceutical regimens based on the discovery of critical regulatory molecules in the differentiation process. With this in mind, we can begin to explore therapeutic options that have never before been available.

Transient expression of type II collagen and tissue mobilization during development of the scleral ossicle, a membranous bone, in the chick embryo.

Development of the chick scleral ossicle was studied with respect to expression of various collagen types, cartilage matrix molecules, and osteoblastic cell surface antigens. The extra-cellular matrix of the scleral ossicle primordium of stage 35.5 chick sclera and the mesenchyme beneath the conjunctival epithelium was immunoreactive with anti-type II collagen antibody, giving the impression that certain materials and/or cell clusters surrounded by reactive matrix were descending from the epithelial-mesenchymal interface to the scleral ossicle primordium. In stage 37 embryos, type II collagen immunoreactivity was restricted to the bone matrix of the scleral ossicles, and persisted through stage 39. However, at stage 41, virtually no type II collagen was detected. In contrast, strong immunostaining of type I collagen was first detected in the developing scleral ossicle at stage 37, coinciding with the formation of mineralized bone matrix. Following the extensive accumulation of type I collagen in bone matrix, type XII collagen was detected at the surface of the bone; both type I and type XII collagen immunostainings then remained. By stage 37, immunoreactivity with a pre-osteoblastic cell surface marker was detected on cells of the scleral ossicle, and typical osteocytes were subsequently identified by both morphological and specific immunostaining techniques. Antibodies other than for type II collagen, specific to chondrogenic mesenchyme or cartilage matrix, never reacted with the scleral ossicle and its primordium during development. Taken together, these observations indicate that the scleral ossicle is a membranous bone, whose development may not require overt chondrogenesis. Implications of type II collagen distribution during the positioning of scleral ossicles and their early bone matrix formation are discussed with respect to the origin and evolution of endoskeletons in vertebrate animals.

In vitro differentiation of bone and hypertrophic cartilage from periosteal-derived cells.

Periosteal cells were enzymatically liberated from the tibiae of young chicks, introduced into cell culture, and allowed to reach confluence. The morphology of the cells gave the impression of a relatively homogeneous population of fibroblast-like cells. These cultured cells did not overtly express osteogenic or chondrogenic properties as judged by their morphology and the lack of reactivity with probes to phenotype-specific antigens of osteoblasts or chondrocytes. The cells were then replated at relatively high density and chronologically evaluated for the differentiation of bone and cartilage. These replated cells formed a multi-layer of fibroblast-like cells, the top portion of which eventually differentiated into bone tissue as evidenced by the presence of mineralization and immunocytochemical reactivity to bone Gla protein- and osteocyte-specific probes. Cells below this distinctive top layer differentiated into chondrocytes, which eventually further developed into hypertrophic chondrocytes as evidenced by their morphology and the presence of immunoreactive type X collagen in the matrix. Mineralization was also observed in the territorial matrix of these hypertrophic chondrocytes, when the culture was augmented with beta-glycerophosphate. Periosteal-derived cells replated at a lower density as controls did not show signs of osteochondrogenic differentiation. These observation suggest that periosteal-derived cells of young chicks contain mesenchymal cells which possess the potential to undergo terminal differentiation into osteogenic or chondrogenic phenotypes depending on local environmental or positional cues.

Immunohistochemical localization of a approximately 66 kD glycosylated phosphoprotein during development of the embryonic chick tibia.

Localization of a approximately 66 kD glycosylated phosphoprotein during morphogenesis of the embryonic chick tibia has been accomplished using immunohistochemistry. Although initial expression of the tibial osteoblast phenotype is detected as early as stage 28.5, with the deposition of osteoid matrix beginning at stage 30, little or no immunoreactivity against the approximately 66 kD glycosylated phosphoprotein is observed in pre-osteoblasts, osteoblasts, osteocytes, or in the uncalcified osteoid matrix during the early events of tibia development. Immunoreactivity was first observed at stage 32 when mineralization of the osteoid matrix is initiated. At this and all later stages, the phosphoprotein is located almost exclusively in the extracellular matrix at the mineralization front with essentially no detectable staining in the adjacent unmineralized osteoid matrix. Similarly, no cellular staining is observed when even the lightly mineralized extracellular matrix is strongly immunoreactive. Only scant immunostaining is present over the heavily mineralized regions, although demineralization of these areas with EDTA exposes a low intensity, punctate staining pattern. Additionally, cryosections of developing calvaria stained with this antiserum only display reactivity in regions of bone matrix undergoing mineralization. These localization studies support the hypothesis that this phosphoprotein is intimately associated with the process of bone matrix mineralization in the developing chick long bone.