Terhi J. Heino

Osteocytes inhibit osteoclastic bone resorption through transforming growth factor-β: Enhancement by estrogen*

Osteocytes are the most abundant cells in bone and distributed throughout the bone matrix. They are connected to the each other and to the cells on the bone surface. Thus, they may also secrete some regulatory factors controlling bone remodeling. Using a newly established osteocyte‐like cell line MLO‐Y4, we have studied the interactions between osteocytes and osteoclasts. We collected the conditioned medium (CM) from MLO‐Y4 cells, and added it into the rat osteoclast cultures. The conditioned medium had no effect on osteoclast number in 24‐h cultures, but it dramatically inhibited resorption. With 5, 10, and 20% CM, there was 25, 39, and 42% inhibition of resorption, respectively. Interestingly, the inhibitory effect was even more pronounced, when MLO‐Y4 cells were pretreated with 10−8 M 17‐β‐estradiol. With 5, 10, and 20% CM, there was 46, 51, and 58% of inhibition. When the conditioned medium was treated with neutralizing antibody against transforming growth factor‐β (TGF‐β), the inhibitory effect was abolished. This suggests that osteocytes secrete significant amounts of TGF‐β, which inhibits bone resorption and is modulated by estrogen. RT–PCR and Western blot analysis show that in MLO‐Y4 cells, the prevalent TGF‐β isoform is TGF‐β3. We conclude that osteocytes have an active, inhibitory role in the regulation of bone resorption. Our results further suggest a novel role for TGF‐β in the regulation of communication between different bone cells and suggest that at least part of the antiresorptive effect of estrogen in bone could be mediated via osteocytes. J. Cell. Biochem. 85: 185–197, 2002.

WNT1 Mutations in Early-Onset Osteoporosis and Osteogenesis Imperfecta

This report identifies human skeletal diseases associated with mutations in WNT1. In 10 family members with dominantly inherited, early-onset osteoporosis, we identified a heterozygous missense mutation in WNT1, c.652T→G (p.Cys218Gly). In a separate family with 2 siblings affected by recessive osteogenesis imperfecta, we identified a homozygous nonsense mutation, c.884C→A, p.Ser295*. In vitro, aberrant forms of the WNT1 protein showed impaired capacity to induce canonical WNT signaling, their target genes, and mineralization. In mice, Wnt1 was clearly expressed in bone marrow, especially in B-cell lineage and hematopoietic progenitors; lineage tracing identified the expression of the gene in a subset of osteocytes, suggesting the presence of altered cross-talk in WNT signaling between the hematopoietic and osteoblastic lineage cells in these diseases.

Differentiation of osteoblasts and osteocytes from mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are multipotent cells that arise from the mesenchyme during development. They reside in the bone marrow close to hematopoietic stem cell niches allowing them to maintain bone marrow homeostasis and to regulate the maturation of both hematopoietic and non-hematopoietic cells. MSCs possess an extensive potential to proliferate and differentiate e.g. into osteoblasts, osteocytes, adipocytes and chondrocytes. Nevertheless, there still are some open questions about the complex process of MSC differentiation involving different transcription factors and signaling pathways, which will be discussed in this review. We also shortly introduce the characteristics and function of bone-forming osteoblasts and their role in angiogenesis. MSCs are of interest in clinical applications, since they can be easily isolated from bone marrow aspirates and expanded in vitro. When the source of osteoprogenitors is compromised, cell-based therapies could provide a novel way to repair bone defects. Indeed, there is an increasing interest in the use of MSCs and more differentiated cells in clinical applications for bone repair, which will be introduced in this review. A major section of the review is dedicated to the functions of osteocytes in the regulation of bone remodeling. Finally, we present an original hypothesis about the possible role of osteocytes in future bone tissue engineering.

Bone Marrow Cell Differentiation Induced by Mechanically Damaged Osteocytes in 3D Gel-Embedded Culture†

Osteocytes are suggested to have a crucial role in the initial resorptive phase of bone turnover after microdamage. To study the role of osteocytes in targeted remodeling, we developed an in vitro model, in which osteocytes can be locally damaged and their interactions with bone marrow cells studied. Our results show that the damaged osteocytes activate the osteoclast precursors by soluble factors and thus can control the initial phase of targeted remodeling.

Circulating plastic adherent mesenchymal stem cells in aged hip fracture patients

We examined the presence of circulating plastic adherent multipotent mesenchymal stem cells (MSCs) in fracture patients. Three patient groups (n = 10–18) were evaluated, including elderly females with a femoral neck fracture treated with cemented hemiarthroplasty, an age‐ and sex‐matched group with hip osteoarthritis (OA) treated with cemented total hip arthroplasty (THA), and younger adults with surgically treated lower extremity fractures. The presence of circulating MSCs pre‐ and postoperatively was compared to bone marrow (BM) MSCs from the same subjects. Criteria for identifying MSCs included cell surface markers (CD105+, CD73+, CD90+, CD45−, CD14−), proliferation through several passages as well as osteogenic, chondrogenic, and adipogenic differentiation. Plastic adherent MSCs were found in peripheral blood (PB) from 22% of hip fracture patients, 46% of younger fracture patients, and in none of 63 pre‐ and postmenopausal women with hip OA. When detectable, circulating MSCs appeared between 39 and 101 h after fracture. PB derived MSCs did not differ from BM derived MSCs, except for a small population (<15%) of CD34+ cells among PB derived MSCs. This initial study indicates mobilization of MSCs into the circulation in response to fracture, even in very old patients, while circulating MSCs were not detectable before or after elective THA.

The novel estrogen receptor G-protein-coupled receptor 30 is expressed in human bone.

Estrogens have significant impact on bone mineral metabolism. Besides the classical estrogen receptors (ERalpha and ERbeta), a trans-membrane G-protein-coupled receptor (GPR30) has been demonstrated to mediate estrogenic effects. We aimed to study whether GPR30 is expressed in bone cells and if so, whether the level of expression is developmentally regulated. Metaphyseal bone biopsies were collected from the tibia in 14 boys and 6 girls, all at different stages of puberty. GPR30 protein expression was studied by immunohistochemistry in paraffin-embedded sections. GPR30-positive osteocytes and osteoblasts were quantified and linear regression analysis was applied. Cytoplasmic GPR30 expression was detected in osteoblasts, osteocytes, and osteoclasts. Osteocytes were more frequently positive for GPR30 than osteoblasts (58+/-4% vs 46+/-3% positive cells respectively, P<0.05). Detailed analysis demonstrated that GPR30 positivity declined during pubertal development in osteocytes (R=-0.56, P<0.01) but not in osteoblasts (R=-0.31, P>0.05). No sex difference was observed in the numbers of GPR30-positive osteoblasts or osteocytes. Furthermore, GPR30 expression did not correlate with chronological or bone age. In conclusion, the novel ER GPR30 is expressed in osteoblasts, osteocytes, and osteoclasts suggesting that non-genomic estrogen signaling via GPR30 may exist in bone. However, the functional role of GPR30 in bone tissue remains to be elucidated.

Evidence for the role of osteocytes in the initiation of targeted remodeling

Microdamage in bone contributes to fractures and acts as a stimulus for bone remodeling. Osteocytes are the most abundant cells in bone, and their death by microdamage has been suggested to be the major event leading in the initiation of osteoclastic bone resorption. Even though there is increasing evidence that osteocyte density, microcracks and targeted remodeling are related, there still exist several questions. For example, how osteoclasts are targeted to the specific site of microdamage for repair. It has been proposed that apoptotic osteocytes could secrete a specific signal to target osteoclasts. The other question is the nature of this signal. To elucidate the role of microdamage-induced osteocyte cell death in the initiation of targeted remodelling, this paper discusses the potential use of an in vitro model, in which osteocytes can be three-dimensionally cultured and locally damaged. Furthermore, the method enables one to study the osteocyte-derived soluble interactions with bone marrow cells. It was demonstrated that damaged osteocytes locally affect osteoclast precursors by secreting osteoclastogenic factors, and thus can have a role in the initiation of resorption in bone remodelling. This strongly supports the idea that damage to osteocyte cellular network has the potential to stimulate osteoclastic proliferation and therefore the activation of Basic Multicellular Units (BMUs).

Osteocyte-derived HB-GAM (pleiotrophin) is associated with bone formation and mechanical loading

HB-GAM (also known as pleiotrophin) is a cell matrix-associated protein that is highly expressed in bone. It affects osteoblast function, and might therefore play a role in bone development and remodeling. We aimed to investigate the role of HB-GAM in bone in vivo and in vitro. The bones of HB-GAM deficient mice with an inbred mouse background were studied by histological, histomorphometrical, radiological, biomechanical and mu-CT analyses and the effect of immobilization was evaluated. HB-GAM localization in vivo was studied. MLO-Y4 osteocytes were subjected to fluid shear stress in vitro, and gene and protein expression were studied by subtractive hybridization, quantitative PCR and Western blot. Human osteoclasts were cultured in the presence of rhHB-GAM and their formation and resorption activities were assayed. In agreement with previous reports, the skeletal structure of the HB-GAM knockout mice developed normally. However, a growth retardation of the weight-bearing bones was observed by 2 months of age, suggesting a link to physical activity. Adult HB-GAM deficient mice were characterized by low bone formation and osteopenia, as well as resistance to immobilization-dependent bone remodeling. HB-GAM was localized around osteocytes and their processes in vivo and furthermore, osteocytic HB-GAM expression was upregulated by mechanical loading in vitro. HB-GAM did not affect on human osteoclast formation or resorption in vitro. Taken together, our results suggest that HB-GAM is an osteocyte-derived factor that could participate in mediating the osteogenic effects of mechanical loading on bone.

Microdamage detection and repair in bone: Fracture mechanics, histology, cell biology

Bone is an elementary component in the human skeleton. It protects vital organs, regulates calcium levels and allows mobility. As a result of daily activities, bones are cyclically strained causing microdamage. This damage, in the form of numerous microcracks, can cause bones to fracture and therefore poses a threat to mechanical integrity. Bone is able to repair the microcracks through a process called remodelling which is tightly regulated by bone forming and resorbing cells. However, the manner by which microcracks are detected, and repair initiated, has not been elucidated until now. Here we show that microcrack accumulation causes damage to the network of cellular processes, resulting in the release of RANKL which stimulates the differentiation of cells specialising in repair.

Comparison of the osteogenic capacity of minipig and human bone marrow-derived mesenchymal stem cells

Minipigs are a recommended large animal model for preclinical testing of human orthopedic implants. Mesenchymal stem cells (MSCs) are the key repair cells in bone healing and implant osseointegration, but the osteogenic capacity of minipig MSCs is incompletely known. The aim of this study was to isolate and characterize minipig bone marrow (BM) and peripheral blood (PB) MSCs in comparison to human BM‐MSCs. BM sample was aspirated from posterior iliac crest of five male Göttingen minipigs (age 15 ± 1 months). PB sample was drawn for isolation of circulating MSCs. MSCs were selected by plastic‐adherence as originally described by Friedenstein. Cell morphology, colony formation, proliferation, surface marker expression, and differentiation were examined. Human BM‐MSCs were isolated and cultured from adult fracture patients (n = 13, age 19–60 years) using identical techniques. MSCs were found in all minipig BM samples, but no circulating MSCs could be detected. Minipig BM‐MSCs had similar morphology, proliferation, and colony formation capacities as human BM‐MSCs. Unexpectedly, minipig BM‐MSCs had a significantly lower ability than human BM‐MSCs to form differentiated and functional osteoblasts. This observation emphasizes the need for species‐specific optimization of MSC culture protocol before direct systematic comparison of MSCs between human and various preclinical large animal models can be made.