Phenotypic and Molecular Heterogeneity in Fibrodysplasia Ossificans Progressiva

Fibrodysplasia (myositis) ossificans progressiva (FOP) is an extremely rare inherited disorder in which progressive ossification of major striated muscles, often following injury, is associated with abnormal skeletal patterning. Altered expression of bone morphogenetic proteins may be a contributory cause. To examine this hypothesis, we compared the patterns of expression of bone morphogenetic proteins (BMPs) mRNAs from lymphoblastoid cell lines from two small multigenerational families with autosomal dominant transmission of FOP. Although affected members of both families showed the characteristic phenotype of FOP, one family was more severely affected than the other. Expression of mRNAs for BMP-1, 2, 3, 5, and 6 mRNAs were not detected within the more severely affected family, but BMP-4 mRNA was expressed in affected but not unaffected members of this family. The results of linkage exclusion analysis using a highly polymorphic microsatellite marker near the BMP-4 gene were consistent with linkage of FOP and BMP-4 in this family. Within the less severely affected family, affected and unaffected members showed similar levels of mRNA expression of BMPs 1, 2, 4, and 5, and linkage of FOP to the BMP-4 gene was excluded. It is concluded that clinical, radiographic, and biochemical data in these two families with FOP establish clinical and molecular heterogeneity and also suggest the possibility of genetic heterogeneity. Received: 22 January 1998 / Accepted: 9 December 1998     

Experimental–Computational Evaluation of Human Bone Marrow Stromal Cell Spreading on Trabecular Bone Structures

The clinical application of macro-porous scaffolds for bone regeneration is significantly affected by the problem of insufficient cell colonization. Given the wide variety of different scaffold structures used for tissue engineering it is essential to derive relationships for cell colonization independent of scaffold architecture. To study cell population spreading on 3D structures decoupled from nutrient limitations, an in vitro culture system was developed consisting of thin slices of human trabecular bone seeded with Human Bone Marrow Stromal Cells, combined with dedicated μCT imaging and computational modeling of cell population spreading. Only the first phase of in vitro scaffold colonization was addressed, in which cells migrate and proliferate up to the stage when the surface of the bone is covered as a monolayer, a critical prerequisite for further tissue formation. The results confirm the model’s ability to represent experimentally observed cell population spreading. The key advantage of the computational model was that by incorporating complex 3D structure, cell behavior can be characterized quantitatively in terms of intrinsic migration parameters, which could potentially be used for predictions on different macro-porous scaffolds subject to additional experimental validation. This type of modeling will prove useful in predicting cell colonization and improving strategies for skeletal tissue engineering.     

Osteoprogenitor response to semi-ordered and random nanotopographies

In bone tissue engineering, it is desirable to use materials to control the differentiation of mesenchymal stem cell populations in order to gain direct bone apposition to implant materials. It has been known for a number of years that microtopography can alter cell adhesion, proliferation and gene expression. More recently, the literature reveals that nanotopography is also of importance. Here, the reaction of primary human osteoprogenitor cell populations to nanotopographies down to 10 nm in size is considered. The topographies were originally produced by colloidal lithography and polymer demixing on silicon and then embossed (through an intermediate nickel shim) into polymethylmethacrylate. The biological testing considered cell morphology (image analysis of cell spreading and scanning electron microscopy), cell cytoskleton and adhesion formation (fluorescent staining of actin, tubulin, vimentin and vinculin) and then subsequent cell growth and differentiation (fluorescent staining of osteocalcin and osteopontin). The results demonstrated that the nanotopographies stimulated the osteoprogenitor cell differentiation towards an osteoblastic phenotype.     

The effect of anisotropic architecture on cell and tissue infiltration into tissue engineering scaffolds

A common phenomenon in tissue engineering is rapid tissue formation on the outer edge of the scaffold which restricts cell penetration and nutrient exchange to the scaffold centre, resulting in a necrotic core. To address this problem, we generated scaffolds with both random and anisotropic open porous architectures to enhance cell and subsequent tissue infiltration throughout the scaffold for applications in bone and cartilage engineering. Hydroxyapatite (HA) and poly(D,L-lactic acid) (P(DL)LA) scaffolds with random open porosity were manufactured, using modified slip-casting and by supercritical fluid processing respectively, and subsequently characterised. An array of porous aligned channels (400 microm) was incorporated into both scaffold types and cell (human osteoblast sarcoma, for HA scaffolds; ovine meniscal fibrochondrocytes, for P(DL)LA scaffolds) and tissue infiltration into these modified scaffolds was assessed in vitro (cell penetration) and in vivo (tissue infiltration; HA scaffolds only). Scaffolds were shown to have an extensive random, open porous structure with an average porosity of 85%. Enhanced cell and tissue penetration was observed both in vitro and in vivo demonstrating that scaffold design alone can influence cell and tissue infiltration into the centre of tissue engineering scaffolds.     

Characterization and multipotentiality of human fetal femur-derived cells: implications for skeletal tissue regeneration

To date, the plasticity, multipotentiality, and characteristics of progenitor cells from fetal skeletal tissue remain poorly defined. This study has examined cell populations from human fetal femurs in comparison with adult-derived mesenchymal cell populations. Real-time quantitative polymerase chain reaction demonstrated expression of mesenchymal progenitor cell markers by fetal-derived cells in comparison with unselected adult-derived and immunoselected STRO-1-enriched adult populations. Multipotentiality was examined using cells derived from femurs and single-cell clones, culture-expanded from explants, and maintained in basal medium prior to exposure to adipogenic, osteogenic, and chondrogenic conditions. Adipocyte formation was confirmed by Oil Red O lipid staining and aP2 immunocytochemistry, with expression of peroxisome proliferation-activated receptor-gamma detected only in adipogenic conditions. In chondrogenic pellets, chondrocytes lodged within lacunae and embedded within dense proteoglycan matrix were observed using Alcian blue/Sirius red staining and type II collagen immunocytochemistry. Osteogenic differentiation was confirmed by alkaline phosphatase staining and type I collagen immunocytochemistry as well as by gene expression of osteopontin and osteocalcin. Single-cell clonal analysis was used to demonstrate multipotentiality of the fetal-derived populations with the formation of adipogenic, chondrogenic, and osteogenic populations. Mineralization and osteoid formation were observed after culture on biomimetic scaffolds with extensive matrix accumulation both in vitro and in vivo after subcutaneous implantation in severely compromised immunodeficient mice. These studies demonstrate the proliferative and multipotential properties of fetal femur-derived cells in comparison with adult-derived cells. Selective differentiation and immunophenotyping will determine the potential of these fetal cells as a unique alternative model and cell source in the restoration of damaged tissue.     

Effects of a surface topography composite with puerariae radix on human STRO-1-positive stem cells

Human skeletal stem cells (STRO-1 positive/STRO-1⁺) respond to different topographical features in various ways. On a flat surface these cells spread and tend to develop a fibroblast-like morphology. On a microgrooved surface enriched skeletal stem cell populations prefer to stretch along the grooves, which affects their cellular structure and differentiation, a phenomenon known as contact guidance. Growth factors, hormones and chemicals can also stimulate cell differentiation. A traditional Chinese medicine, puerariae radix, has previously been observed to stimulate bone formation. The active ingredients have been identified as isoflavones with estrogen-like bioactivity. This study combined the effects of microgrooved topology and hormone-like isoflavones in the biodegradable polymer polycaprolactone (PCL). Human osteogenic cells (STRO-1⁺) were cultured on flat PCL, grooved PCL and puerariae powder-impregnated grooved PCL for 5 weeks. Coomassie staining indicated that cell growth and survival was similar on flat PCL, grooved PCL and grooved PCL impregnated with 1 wt.% or 2 wt.% puerariae powder. Grooved PCL impregnated with 2 wt.% puerariae powder was observed to have an influence on protein expression, as observed by positive osteocalcin staining. Protein expression profiles were analyzed by difference gel electrophoresis to identify proteins that showed modulation of expression in response to these different environments. Overall, our results suggest that puerariae powder has an additive effect, along with microgrooved topographical stimulation, to promote changes in the STRO-1⁺ proteome that affect cell phenotype.     

Biomimetic collagen scaffolds for human bone cell growth and differentiation

Type I collagen provides a structural framework for connective tissues and plays a central role in the temporal cascade of events leading to the formation of new bone from progenitors. The aim of this study was to examine the ability of the cell-binding domain of type I collagen (P-15 peptide) to promote human bone marrow stromal cell adhesion, proliferation, and differentiation on three-dimensional scaffolds. Human bone marrow stromal cells were selected, expanded, and cultured on particulate microporous ABM ("pure" hydroxyapatite) phase adsorbed with or without P-15 under basal or osteogenic conditions. Immobilized P-15 increased alkaline phosphatase activity and bone morphogenetic protein 2 (BMP-2) gene expression after 1 and 5 days as determined by real-time polymerase chain reaction. P-15 promoted human bone marrow stromal cell attachment, spreading, and alignment on ABM as well as alkaline phosphatase-specific activity in basal and osteogenic cultures. The presence of mineralized bone matrix, extensive cell ingrowth, and cellular bridging between three-dimensional matrices adsorbed with P-15 was confirmed by confocal microscopy, scanning electron microscopy, and alizarin red staining. Negligible cell growth was observed on ABM alone. In vivo diffusion chamber studies using MF1-nu/nu mice showed bone matrix formation and organized collagen formation after 6 weeks. These studies indicate the potential of P-15 to generate appropriate biomimetic microenvironments for osteoblasts and demonstrate the potential for the exploitation of extracellular matrix cues for osteogenesis and, ultimately, bone regeneration.     

Growth factors for skeletal reconstruction and fracture repair

The demographic challenges of an ageing population have emphasized the need for processes to augment and repair skeletal tissue loss as a consequence of trauma and/or degeneration. A number of bone growth factors have been shown to be expressed during the course of fracture healing, suggesting a potential role in bone and cartilage formation, and in fracture repair. This review focuses on a select number of these growth factors currently under preclinical and clinical evaluation for skeletal regeneration and fracture repair. The limitations in the use of these skeletal factors to augment bone growth, thus improving quality-of-life and reducing the significant social and economic costs associated with skeletal trauma/loss are also considered.     

Acute effects of the Bowman-Birk protease inhibitor in mice

The soybean-derived Bowman-Birk inhibitor (BBI) has been shown to inhibit carcinogenesis in both in vitro and in vivo model systems. In the present study, protease enzyme activity in selected tissues of male strain A mice was measured by hydrolysis of the synthetic substrate Boc-Val-Pro-Arg-MCA (t-butoxycarbornylvalylprolylarginine 7-amido-4-methylcoumarin). When added to homogenates of lung, liver and kidney in vitro, purified BBI inhibited hydrolytic activity at concentrations ranging from 10-100 microM. In vivo, hydrolytic activity was found to be significantly and in a dose-dependent manner, decreased in the lung as early as 2 h after i.p. injection of purified BBI. Less inhibition was found in the liver and kidney after in vivo administration of purified BBI. A crude preparation of BBI, given at 100 mg/kg, had no influence on the overall disposition of radiolabeled benzo[alpha]pyrene in mice although it decreased the hepatic activities of cytochrome P-450, 7-ethoxycoumarin-O-deethylase and ethoxy resorufin-O-deethylase. It is concluded that the chemopreventive effects of BBI on mouse lung tumor development are most likely mediated through its protease-inhibitory properties in the target organ rather than by a non-specific effect on metabolism and disposition of carcinogens.     

Inhibitory effects of the bone-derived growth factors osteoinductive factor and transforming growth factor-beta on isolated osteoclasts.

Demineralized bone matrix contains a number of growth factors for osteoblast-like cells. Two of these, the novel glycoprotein osteoinductive factor (OIF) and transforming growth factor-beta (TGF beta), act together to cause ectopic bone formation in vivo. Since OIF, like TGF beta, is likely released from bone when the matrix is resorbed, we examined the effects of homogeneous OIF and TGF beta on osteoclast function. Osteoclast function was tested in isolated avian osteoclasts and was measured in terms of tartrate-resistant acid phosphatase (TRAP) activity, oxygen-derived free radical production, and formation of characteristic resorption lacunae on slices of sperm whale dentine. OIF (50-100 ng/ml) inhibited the capacity of these osteoclasts to form lacunae whether assessed by the number of excavations per slice or by the total area resorbed. OIF (10-100 ng/ml) or TGF beta (10-20 ng/ml) caused a decrease in TRAP activity as well as a reduction in oxygen-derived free radical generation detected by nitroblue tetrazolium staining. TGF beta had no effect on the resorption capacity of isolated osteoclasts in concentrations that inhibited TRAP activity and nitroblue tetrazolium staining. These results suggest that growth regulatory factors, such as OIF and TGF beta, released during the resorption of bone may be endogenous inhibitors of continued osteoclastic activity. This cessation of osteoclast activity may be an essential preliminary step to the new bone formation that occurs at resorption sites during bone remodeling.