The cellular and molecular mechanisms that underlie age-dependent osteoporosis, the most common disease in the Western Hemisphere, are poorly understood in part due to the lack of appropriate animal models in which to study disease progression. Here, we present a model that shows many similarities to the human disease. Sca-1, well known for its expression on hematopoietic stem cells, is present on a subset of bone marrow stromal cells, which potentially include mesenchymal stem cells. Longitudinal studies showed that Sca-1 ؊/؊ mice undergo normal bone development but with age exhibit dramatically decreased bone mass resulting in brittle bones. In vivo and in vitro analyses demonstrated that Sca-1 is required directly for the self-renewal of mesenchymal progenitors and indirectly for the regulation of osteoclast differentiation. Thus, defective mesenchymal stem or progenitor cell self-renewal may represent a previously uncharacterized mechanism of age-dependent osteoporosis in humans.O steoporosis is a multifactorial, age-related metabolic bone disease characterized by low bone mineral density (BMD) and the deterioration of the microarchitecture of cancellous bone, leading to enhanced bone fragility and increased risk of fracture (1). Type I osteoporosis, also called postmenopausal osteoporosis because it primarily affects postmenopausal women, is characterized by increased bone turnover and accelerated cancellous bone loss, increasing the risk of vertebral fracture. Type II osteoporosis, or age-related͞dependent osteoporosis, affects older women and men and its origins are far less understood than postmenopausal osteoporosis. Although type II osteoporosis is not accompanied by increased bone turnover, it leads to increased risk of hip fracture and has a greater mortality and morbidity than type I osteoporosis.Type I and II osteoporosis are thought to result from dysregulated bone remodeling during aging. Normal bone remodeling requires an exquisite balance between bone resorption by osteoclasts and bone formation by osteoblasts. Osteoblasts are mesenchymal cells that originate from a multipotential precursor, often referred to as the mesenchymal stem cell (MSC), which also gives rise to other lineages such as adipocytes, chondrocytes, and muscle (2, 3). By contrast, osteoclasts differentiate from hematopoietic monocyte͞macrophage precursors (4).Stem cell antigen 1 (Sca-1; also known as Ly-6A) is an 18-kDa glycosyl phosphatidylinositol-anchored cell surface protein of the Ly-6 gene family expressed by hematopoietic stem cells (HSCs), skeletal muscle stem cells, mammary epithelial stem cells, subsets of hematopoietic progenitors, lymphocytes and macrophages, and a subpopulation of bone marrow (BM) stromal cells including osteoblasts (5-11). Sca-1 Ϫ/Ϫ mice exhibit defects in T cell signaling (12) and HSC self-renewal (13).Here, we report that Sca-1 Ϫ/Ϫ mice also model human agerelated (type II) osteoporosis with reduced BMD and increased susceptibility to fractures. Unlike type I osteoporosis, which results from an...

Successful joint resurfacing by tissue-engineered cartilage has been limited, in part, by an inability to secure the implant to bone. To overcome this, we have developed the methodology to form a cartilage implant in vitro consisting of a layer of cartilagenous tissue overlying a porous, biodegradable calcium polyphosphate (CPP) substrate. As bone will grow into the CPP after implantation, it will result in anchorage of the cartilage. In this study, the cartilagenous tissue formed in vitro after 8 weeks in culture was characterized and compared to native articular cartilage. Light microscopic examination of histological sections showed that there was a continuous layer of cartilagenous tissue on, and integrated with the subsurface of, the CPP substrate. The in vitro-formed tissue achieved a similar thickness to native articular cartilage (mean +/- SEM: in vitro = 0.94 +/- 0.03 mm; ex vivo = 1.03 +/- 0.01 mm). The cells in the in vitro-formed tissue synthesized large proteoglycans (Kav +/- SEM: in vitro = 0.27 +/- 0.01; ex vivo = 0.27 +/- 0.01) and type II collagen similar to the chondrocytes in the ex-vivo cartilage. The in vitro-formed tissue had a similar amount of proteoglycan (GAG microg/mg dry wt.: in vitro = 198 +/- 10; ex vivo = 201 +/- 13) but less collagen than the native cartilage (hydroxyproline microg/mg dry wt.: in vitro = 21 +/- 1; ex vivo = 70 +/- 8). The in vitro-formed tissue had only about 3% of the load-bearing capacity and stiffness of the native articular cartilage, determined from unconfined mechanical compression testing. Although low, this was within the range of properties reported by others for tissue-engineered cartilage. It is possible that the limited load-bearing capacity is the result of the low collagen content and further studies are required to identify the conditions that will increase collagen synthesis.

The formation of cartilaginous tissue in vitro is a promising alternative to repair damaged articular cartilage. However, recent attempts to tissue-engineer articular cartilage that has similar properties to the native tissue have proven to be difficult. The in vitroformed cartilaginous tissue typically has a similar proteoglycan content to native cartilage, but has a reduced collagen content and only a fraction of the mechanical properties. In this study, we investigated whether the intermittent application of cyclic shearing forces during tissue formation would improve the tissue quality. Chondrocyte cultures were stimulated a t a 2'%1 shear strain amplitude at a frequency of 1 Hz for 400 cycles every 2nd day. At one week, both collagen and proteoglycan synthesis increased (23 f 6% and 20 f 6'%1, respectively) over the unstimulated, static controls. At four weeks, an increased amount of tissue formed . Tissues that formed in the presence of shearing forces also displayed a 3-fold increase in compressive load-bearing capacity (stirnulatrri: 16 f 5, unstitnuluted: 5 f I kPa max. equilibrium stress) and a 6-fold increase in stiffness (stirnuluted 112 f 36, unstirnulutc'cl: 20 f 6 kPa max. equilibrium modulus) compared to the static controls. These results demonstrate that intermittent application of dynamic shearing forces over a four-week period improves the quality of cartilaginous tissue formed in vitro. Interestingly, low amplitudes of shear stimulation for short periods of time (6 min of stimulation applied every 2nd day) produced these changes.

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Topographical substrates, controlling cell adhesion in two and three dimensions, are reviewed and compared with two- and three-dimensional models.

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A single application of cyclic loading to chondrocytes early in culture increased matrix accumulation and enhanced the mechanical properties of the in vitro-formed tissue. This suggests that mechanical forces do not have to be applied intermittently over long periods of time to accelerate in vitro tissue formation.

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