The rapidly expanding field of skeletal biology promises to yield major treatment advances in many areas of orthopaedic surgery over the next few decades. Our understanding of skeletal repair, skeletal tissue engineering, and normal and abnormal bone metabolism have already greatly improved with the application of new techniques for the study of cellular and molecular biology to skeletal tissues. The Brigham and Women's Skeletal Biology Laboratory and the Massachusetts General Hospital Skeletal Biology Research Center have particular interest in the study of metabolic bone disease, skeletal tissue engineering, and osteoinductive materials. Some of our recent work is outlines in this paper.

Metabolic Bone Disease

          Osteoporosis-related fractures are epidemic in the elderly, particularly in women whose loss of bone accelerates after menopause. Improved understanding of the processes that contribute to the development of osteoporosis is likely to disclose opportunities for novel interventions for this major public health problem. In collaborative studies with clinicians (including Dr. Meryl LeBoff, Director of the BWH Osteoporosis and Skeletal Health Program, and Dr. John Wright, BWH Department of Orthopedic Surgery) and in basic laboratory work, we have made a number of pertinent observations. Although the influence of menopause on bone is well appreciated, age-related loss of bone may be also related to nutrition, diminished adrenal function (the adrenopause), and altered patterns of differentiation of human marrow cells.

Vitamin D Deficiency is Common in Community-Dwelling Women with Hip Fractures
          Bone marrow samples were collected from 29 post-menopausal women living at home and presenting with hip fractures that required arthroplasty, and from 53 women with osteoarthritis of the hip scheduled for elective total hip arthroplasty (THA). We found that women with hip fractures were more likely than age-matched controls undergoing elective total hip arthroplasty to have vitamin D deficiency (52% vs. 9%) and secondary hyperparathyroidism (66% vs. 8%).1 Vitamin D deficiency is preventable and treatable, but community-dwelling women are rarely suspected of being vitamin D-deficient and patients with hip fractures are not routinely tested for the condition. If orthopedic surgeons identified and treated vitamin D-deficiency in hip fracture patients, the ensuing normalization of parathyroid hormone levels may facilitate fracture healing and reduce the risk of future fractures. This hypothesis is being tested in a clinical study at Brigham and Women's Hospital.

Osteoporosis Occurs in Osteoarthritic Post-Menopausal Women Scheduled for Elective Total Hip Arthroplasty
          A large percentage of osteoarthritic women scheduled for elective total hip replacement was found to be at substantial risk for osteoporosis-related fractures. This was unexpected because of literature suggesting an inverse relationship between osteoarthritis and osteoporosis.2 Twelve of 53 women (23%) undergoing elective total hip arthroplasty had bone mineral density (BMD) low enough to meet the World Health Organization's definition of osteoporosis (T score < -2.5).1 The age-adjusted Z-scores for BMD in these 12 women were equivalent to those for the women who presented with hip fractures (Figure 1). Six of the 12 women had vitamin D-deficiency and three had elevated parathyroid hormone levels. These findings suggest that routine assessment of bone density and vitamin D status may be warranted in older postmenopausal women seen in orthopaedic practice.

The Adrenopause Contributes to Age-Related Changes in Bone Metabolism
          The consequences of the menopause on skeletal health are well appreciated. Less is known about the effects of the adrenopause (the decline in adrenal secretion of the steroid dehydroepiandrosterone sulfate, DHEAS) on skeletal metabolism. We studied 102 healthy women ranging from 30-year-old premenopausal women to estrogen-deficient postmenopausal women up to 95 years of age and found that circulating levels of DHEAS decreased sharply with age (r= -0.52, p <0.0001).3 IGF-I levels also decreased with age (r = -0.49, p<0.00001), whereas levels of IL-6 increased, and both trends were correlated with DHEAS levels (r = 0.43. and r = -0.32, respectively). These findings are consistent with results from other populations.4,5 (Figure 2) Because DHEAS acts to stimulate the anabolic factor IGF-1 and inhibit the catabolic factor IL-6, we propose that the age-related decrease in DHEAS contributes to skeletal aging. (Figure 3) Clinical studies are underway to determine the effect of DHEAS replacement therapy on markers of bone turnover and bone mineral density. In addition, in vitro studies examine the effects of DHEAS on IGF-I and IL-6 production by aged human cells.

The Role of Bone Marrow in Skeletal Aging
          Increased Osteoclastic Differentiation With Age
          Analysis of bone marrow obtained at the time of elective total hip arthroplasty revealed an age-related increase in formation of osteoclasts from progenitor/precursor cells.6 In addition, marrow from women receiving estrogen replacement therapy at the time of total hip arthroplasty generated fewer osteoclasts than did marrow from unsupplemented women. Analysis of cultured marrow cells established an age-related increase in the production of bone-resorbing cytokines such as IL-6 and IL-11 in marrow cells.7 This trend was suppressed in patients receiving estrogen replacement therapy.7 These data suggest that the age-related increase in marrow-derived osteoclasts results from increased marrow production of interleukins. They also indicate that some of the beneficial effects of estrogen replacement therapy may result from suppression of the production of these interleukins. Current research is aimed to determine whether other anti-osteolytic agents (such as DHEAS and bisphosphonates) suppress resorptive cytokines. In addition, estrogen status is now routinely recorded for all patients entered into the BWH Total Joint Registry so that the influence of estrogen replacement therapy on the results of total joint arthroplasty can be analyzed in future studies.

          Decreased Osteoblastic Differentiation With Age
          We measured the potential of human marrow cells to differentiate into osteoblasts in conventional 2-dimensional cell culture. Analysis of 28 samples of marrow from men between 37 to 80 years of age showed an age-related decline in differentiation of osteoblasts (r = -0.48, p<0.01).8 These data are consistent with our hypothesis that skeletal aging is a consequence of aging of the marrow. (Figure 5) With age, human marrow stromal cells appear to be less capable of becoming bone-forming cells, and more capable of supporting differentiation of bone-resorbing cells. Continuing studies evaluate agents with the potential to prevent these age-related changes, such as DHEA, estrogens, and androgens.

Skeletal Tissue Engineering

           The development of three-dimensional culture techniques has made it possible to model many in vivo processes more accurately in the laboratory. The ability to grow tissues of chosen shape and composition may ultimately provide novel treatments with engineered skeletal tissue. In collaborative studies with Dr. Thomas Minas (BWH Department of Orthopaedic Surgery), the integration of cartilage grown on novel biocompatible three-dimensional devices with articular cartilage is being studied in animals.

          The skeletal biology laboratories at BWH and MGH have developed improved techniques for growing skeletal tissues in three-dimensional cultures; are using engineered tissues to study the transduction of biophysical signals in cartilage; and are developing techniques for engineering bone and joints.

Three-Dimensional Culture Techniques
          Three-dimensional tissue cultures are created by growing cells on a scaffold resembling a sponge. Ongoing research seeks to define the advantages and disadvantages of various materials for use as a scaffold. For example, resorbable material may be useful unless products of their resorption affect cell growth and function.
We have shown that perfusion of medium is important in many three-dimensional cultures. Perfusion is required for mouse bone marrow cells to remain viable and to continue hemnatopoiesis in three-dimensional cultures.9 Perfusion and hydrostatic pressure also enhance chondrogenesis10 and osteogenesis.11

Engineered Cartilage Tissue
          Tissue grown in three-dimensional culture is useful in the study of mechanisms by which physical stimuli modulate the organization and metabolic activity of cells. Dr. Shuichi Mizuno is seeking to determine how biophysical factors such as hydrostatic fluid pressure and microstrain are translated into intracellular signals in chondrocytes. In particular, the roles of cellular morphology (e.g. the cytoskeleton) and other mediators (such as calcium flux, pH changes, and nitric oxide) are being investigated with fluorescent tracers by two-photon confocal microscopy.

Engineered Bone
          The three-dimensional culture technique has been used as a scaffold for bone formation by osteoblast progenitor cells present in human bone marrow. Experiments measuring the expression of markers of bone formation in engineered bone show a decline in osteoblast activity with increasing age of the subject (r = -0.48, p < 0.01).8 This culture system will help test the feasibility of engineering of human bone tissue from marrow cells.

Engineered Joints
          The engineering of growing joints for potential application in pediatric orthopedic conditions represents a far more complex situation. Drs. David Zaleske, Glowacki, and Mizuno have proposed that a devitalized joint anlagen from one species may serve as a morphogenic scaffold for chondrocytes from another species. As a feasibility project, murine cells are seeded into a devitalized chick joint anlagen and the construct is transplanted into a compatible neonatal mouse. As critical first steps, it is important to gain an understanding of the influence of the developmental age of the anlagen and to determine the optimal method for seeding the anlagen with cells. Before clinical applications can be realized, we will test the efficacy of the technique in a large animal model and assess the growth potential of the chimeric joint. Ultimately, it may be possible to produce synthetic anlagen.

Osteoinduction

           Observations in humans12 and animals13 show that demineralized bone implants can induce heterotopic bone formation in soft tissue sites. We are investigating the genetic and epigenetic mechanisms of this event and have shown that human skin fibroblasts can be induced to produce cartilage matrix in three-dimensional cultures packed with demineralized bone powder.14 Dr. Karen Yates is identifying the master genes that regulate this differentiation. We are also testing the hypothesis that such cultured tissue may be manipulated to be maintained in vivo as cartilage or to undergo endochondral ossification when needed. Understanding of these mechanisms is of fundamental biological interest, but is also likely to lead to the synthesis of clinically useful materials that can induce the formation of skeletal tissues.

The Development of Novel Osteoinductive Implants
          The development of osteoinductive implants for clinical use has proved complex. The development of scaffolds for three-dimensional culture was a major advance, but our work has demonstrated that some bioresorbable polymers inhibit osteoinduction and, in fact, interfere with bone formation in healing wounds.15 Similarly, some readily soluble forms of hydroxyapatite are incompatible with osteoinduction.16 It has become apparent that composite bone substitutes must be more than osteoinductive and osteoconductive; they must also be osteocompatible. We are using novel small animal models to screen potential implants for each of these properties.

Identification of New Osteoinductive Factors
          The discovery and development of osteoinductive factors has been hampered by the need to perform cumbersome in vivo assays in order to demonstrate heterotopic chondrogenesis and/or osteogenesis. Use of the three-dimensional culture approach as a screening assay should accelerate discovery of new osteoinductive factors.

Conclusion

           As this review of our recent work shows, improved understanding of the molecular and cellular biology of musculoskeletal tissues can influence the treatment of patients in important ways. Skeletal biology has become an integral part of orthopedic research at Harvard.

Julie Glowacki, PhD is the Director of Skeletal Biology in the Department of Orthopaedic Surgery at Brigham and Women's Hospital and Associate Professor of Orthopaedic Surgery at Harvard Medical School. She is also Director of the MGH Skeletal Biology Research Center at Massachusetts General Hospital and Associate Professor of Oral and Maxillofacial Surgery at Harvard Medical School.

Address correspondence to:
Julie Glowacki, PhD; Orthopedic Research; 75 Francis St.; Boston, MA 02115.
e-mail: Glowacki.Julie@MGH.Harvard.edu