Decorin (dcn) and biglycan (bgn), two members of the family of small leucine-rich proteoglycans (SLRPs), are the predominant proteoglycans expressed in skin and bone, respectively. Targeted disruption of the dcn gene results in skin laxity and fragility, whereas disruption of the bgn gene results in reduced skeletal growth and bone mass leading to generalized osteopenia, particularly in older animals. Here, we report that bgn deficiency leads to structural abnormality in collagen fibrils in bone, dermis, and tendon, and to a "subclinical" cutaneous phenotype with thinning of the dermis but without overt skin fragility. A comparative ultrastructural study of different tissues from bgn-and dcn-deficient mice revealed that bgn and dcn deficiency have similar effects on collagen fibril structure in the dermis but not in bone. Ultrastructural and phenotypic analysis of newly generated bgn/dcn double-knockout (KO) mice revealed that the effects of dcn and bgn deficiency are additive in the dermis and synergistic in bone. Severe skin fragility and marked osteopenia characterize the phenotype of double-KO animals in which progeroid changes are observed also in the skin.

We cultured MSCs on an ECM made by bone marrow cells to attempt to reconstitute the MSC niche. This ECM promoted replication of mesenchymal progenitors and retention of their multipotentiality. We conclude that the marrow ECM facilitates expansion of mesenchymal progenitors and hypothesize that it plays an important role in the maintenance of MSC stemness.Introduction: Mesenchymal colony-forming cells of the bone marrow comprise mesenchymal stem cells (MSCs) and their transit amplifying progeny, which we term mesenchymal colony-forming units (MCFUs). These progenitors undergo self-renewal and can differentiate into many different cell types including osteoblasts. However, they lose their unique properties when cultured on tissue culture plastic. This indicates that a critical feature of the marrow microenvironment that facilitates retention of stem cell properties is missing in such culture systems. In other tissues, the extracellular matrix (ECM) forms part of the specialized niche that controls stem cell behavior. Therefore, we examined whether a marrow cell-derived ECM promotes retention of the stem cell characteristics of MCFUs in vitro. Materials and Methods: A cell-free ECM was prepared from cultured murine marrow adherent cells. The replication and multipotentiality of murine MCFUs maintained on this marrow cell-derived ECM were examined in vitro and in vivo and compared with the behavior of MCFUs maintained on plastic. Results: The marrow cell-derived ECM was made up of collagen types I, III, and V, syndecan-1, perlecan, fibronectin, laminin, biglycan, and decorin, similar to the composition of the marrow ECM. This ECM preparation promoted MCFU replication, restrained their "spontaneous" differentiation toward the osteoblast lineage, and preserved their ability to differentiate into osteoblasts or adipocytes. Moreover, transplantation of MCFUs expanded on the marrow cell-derived ECM into immunocompromised mice generated five times more bone and eight times more hematopoietic marrow compared with MCFUs expanded on plastic. Conclusions:The marrow ECM facilitates expansion of MCFUs in vitro while preserving their stem cell properties. We hypothesize that the ECM made by bone marrow cells plays an important role in the maintenance of MSC function.

Biglycan (bgn) is a small leucine-rich proteoglycan enriched in extracellular matrices of skeletal tissues. Bgn-deficient mice develop age-related osteopenia with a phenotype that resembles osteoporosis and premature arthritis. In the present study, we have examined the differentiation of bgn-deficient osteoblasts from neonatal murine calvariae and found that the absence of bgn caused less BMP-4 binding, which reduced the sensitivity of osteoblasts to BMP-4 stimulation. The loss of sensitivity resulted in a reduction of Cbfa1 expression, which ultimately led to a defect in the differentiation of osteoblasts. However, the response of bgn-deficient osteoblasts to BMP-4 was completely rescued by reintroduction of biglycan by viral transfection. We propose that biglycan modulates BMP-4-induced signaling to control osteoblast differentiation.

Extracellular matrix glycoproteins and proteoglycans bind a variety of growth factors and cytokines thereby regulating matrix assembly as well as bone formation. However, little is known about the mechanisms by which extracellular matrix molecules modulate osteogenic stem cells and bone formation. Using mice deficient in two members of the small leucine-rich proteoglycans, biglycan and decorin, we uncovered a role for these two extracellular matrix proteoglycans in modulating bone formation from bone marrow stromal cells. Our studies showed that the absence of the critical transforming growth factor-␤ (TGF-␤)-binding proteoglycans, biglycan and decorin, prevents TGF-␤ from proper sequestration within the extracellular matrix. The excess TGF-␤ directly binds to its receptors on bone marrow stromal cells and overactivates its signaling transduction pathway. Overall, the predominant effect of the increased TGF-␤ signaling in bgn/dcn-deficient bone marrow stromal cells is a "switch in fate" from growth to apoptosis, leading to decreased numbers of osteoprogenitor cells and subsequently reduced bone formation. Thus, biglycan and decorin appear to be essential for maintaining an appropriate number of mature osteoblasts by modulating the proliferation and survival of bone marrow stromal cells. These findings underscore the importance of the micro-environment in controlling the fate of adult stem cells and reveal a novel cellular and molecular basis for the physiological and pathological control of bone mass.The extracellular matrix (ECM) 1 provides structural strength to tissues, maintains the shape of organs, and is often involved directly or indirectly in regulating cell proliferation and differentiation (1-3). ECM components modulate the bioactivities of growth factors and cytokines, such as TGF-␤, tumor necrosis factor-␣, and platelet-derived growth factor, by 1) activating them by proteolytic processing (4, 5), 2) inactivating them by sequestering and preventing binding to their respective receptors (6 -9), or 3) directly binding to cytokine receptors, such as the epidermal growth factor receptor (10, 11).Proteoglycans, which are characterized by a core protein with at least one glycosaminoglycan chain attached, commonly mediate the interactions of ECM components with growth factors and cytokines (12). Small leucine-rich proteoglycans (SLRPs) are some of the major non-collagen components of the ECM (13). The core proteins of the SLRPs consist of leucinerich repeats flanked by two cysteine-rich clusters. The size of the core proteins (ϳ40 kDa) is relatively small compared with aggrecan and versican (Ͼ200 kDa) (1, 10, 14). The SLRP superfamily currently consists of 13 known members that can be divided into 3 distinct subfamilies based on the genomic organization, structure, and similarity of their amino acid sequences (13). SLRPs are involved in skeletal growth (15-17), craniofacial structure (15), dentin formation (18), and collagen fibrillogenesis (17,19,20). However, to date, little is known about the precise mec...

The diffi culty in long-term expansion of mesenchymal stem cells (MSCs) using standard culture systems without the loss of their stem cell properties suggests that a critical feature of their microenvironment necessary for retention of stem cell properties is absent in these culture systems. We report here the reconstitution of a native extracellular matrix (ECM) made by human marrow cells ex vivo, which consists of at least collagen types I and III, fi bronectin, small leucine-rich proteoglycans such as biglycan and decorin, and major components of basement membrane such as the large molecular weight proteoglycan perlecan and laminin. Expansion of human MSCs on this ECM strongly promoted their proliferation, retained their stem cell properties with a low level of reactive oxygen species (ROS), and substantially increased their response to BMP-2. The quality of the expanded cells following each passage was further tested by an in vivo transplantation assay. The results showed that MSCs expanded on the ECM for multiple passages still retained the same capacity for skeletogenesis. In contrast, the bone formation capacity of cells expanded on plastic was dramatically diminished after 6-7 passages. These fi ndings suggest that the marrow stromal cell-derived ECM is a promising matrix for expanding largescale highly functional MSCs for eventual use in stem cell-based therapy. Moreover, this system should also be invaluable for establishment of a unique tissue-specifi c ECM, which will facilitate control of the fate of MSCs for therapeutic applications.

Caspase-3 is a critical enzyme for apoptosis and cell survival. Here we report delayed ossification and decreased bone mineral density in caspase-3-deficient (Casp3 -/-and Casp3 +/-) mice due to an attenuated osteogenic differentiation of bone marrow stromal stem cells (BMSSCs). The mechanism involved in the impaired differentiation of BMSSCs is due, at least partially, to the overactivated TGF-β/Smad2 signaling pathway and the upregulated expressions of p53 and p21 along with the downregulated expressions of Cdk2 and Cdc2, and ultimately increased replicative senescence. In addition, the overactivated TGF-β/Smad2 signaling may result in the compromised Runx2/Cbfa1 expression in preosteoblasts. Furthermore, we demonstrate that caspase-3 inhibitor, a potential agent for clinical treatment of human diseases, caused accelerated bone loss in ovariectomized mice, which is also associated with the overactivated TGF-β/Smad2 signaling in BMSSCs. This study demonstrates that caspase-3 is crucial for the differentiation of BMSSCs by influencing TGF-β/Smad2 pathway and cell cycle progression.

Biglycan is a Class I Small Leucine Rich Proteoglycans (SLRP) that is localized on human chromosome Xq28-ter. The conserved nature of its intron-exon structure and protein coding sequence compared to decorin (another Class I SLRP) indicates the two genes may have arisen from gene duplication. Biglycan contains two chondroitin sulfate glycosaminoglycan (GAG) chains attached near its NH(2) terminus making it different from decorin that has only one GAG chain. To determine the functions of biglycan in vivo, transgenic mice were developed that were deficient in the production of the protein (knockout). These mice acquire diminished bone mass progressively with age. Double tetracycline-calcein labeling revealed that the biglycan deficient mice are defective in their capacity to form bone. Based on this observation, we tested the hypothesis that the osteoporosis-like phenotype is due to defects in cells critical to the process of bone formation. Our data shows that biglycan deficient mice have diminished capacity to produce marrow stromal cells, the bone cell precursors, and that this deficiency increases with age. The cells also have reduced response to tranforming growth factor-beta (TGF-beta), reduced collagen synthesis and relatively more apoptosis than cells from normal littermates. In addition, calvaria cells isolated from biglycan deficient mice have reduced expression of late differentiation markers such as bone sialoprotein and osteocalcin and diminished ability to accumulate calcium judged by alizerin red staining. We propose that any one of these defects in osteogenic cells alone, or in combination, could contribute to the osteoporosis observed in the biglycan knockout mice. Other data suggests there is a functional relationship between biglycan and bone morphogenic protein-2/4 (BMP 2/4) action in controlling skeletal cell differentiation. In order to test the hypothesis that functional compensation can occur between SLRPs, we created mice deficient in biglycan and decorin. Decorin deficient mice have normal bone mass while the double biglycan/decorin knockout mice have more severe osteopenia than the single biglycan indicating redundancy in SLRP function in bone tissue. To further determine whether compensation could occur between different classes of SLRPs, mice were generated that are deficient in both biglycan (class I) and fibromodulin, a class II SLRP highly expressed in mineralizing tissue. These doubly deficient mice had an impaired gait, ectopic calcification of tendons and premature osteoarthritis. Transmission electron microscopy analysis showed that like the decorin and biglycan knockouts, they have severely disturbed collagen fibril structures. Biomechanical analysis of the affected tendons showed they were weaker compared to control animals leading to the conclusion that instability of the joints could be the primary cause of all the skeletal defects observed in the fibromodulin/biglycan knockout mice. These studies present important new animal models for musculoskeletal diseases and provide th...

Joint injury results in cartilage lesions that are characterized by a poor repair response. Adult stem cells are immensely appealing for biological joint repair, such as cartilage tissue engineering and regeneration. However, adult stem cells gradually lose their stemness once they are removed from their in vivo niche for plating in plastic flasks. We utilized a tissue-specific stem cell, synovium-derived stem cell (SDSC), as a model to reconstruct an in vitro three-dimensional stem cell niche. After seeding on SDSC-derived extracellular matrix, the initially wide and flat SDSCs became thin and spindle shaped and were arranged in a three-dimensional configuration with typical stem cell phenotypes. A dramatic increase in cell number and a greatly enhanced chondrogenic capacity were observed, though surprisingly the extracellular matrix-treated SDSCs did not display concomitantly improved adipogenic or osteogenic potentials. Thus, we conclude that a tissue-specific stem cell can be used to prepare its own in vitro niche for stem cell proliferation while maintaining and enhancing its lineage-specific stemness. The ability to reconstitute the in vitro stem cell niche will greatly benefit SDSC-based therapy for cartilage defects.