Mahesh H. Mankani

MT1-MMP-Deficient Mice Develop Dwarfism, Osteopenia, Arthritis, and Connective Tissue Disease due to Inadequate Collagen Turnover

MT1-MMP is a membrane-bound matrix metalloproteinase (MT-MMP) capable of mediating pericellular proteolysis of extracellular matrix components. MT1-MMP is therefore thought to be an important molecular tool for cellular remodeling of the surrounding matrix. To establish the biological role of this membrane proteinase we generated MT1-MMP-deficient mice by gene targeting. MT1-MMP deficiency causes craniofacial dysmorphism, arthritis, osteopenia, dwarfism, and fibrosis of soft tissues due to ablation of a collagenolytic activity that is essential for modeling of skeletal and extraskeletal connective tissues. Our findings demonstrate the pivotal function of MT1-MMP in connective tissue metabolism, and illustrate that modeling of the soft connective tissue matrix by resident cells is essential for the development and maintenance of the hard tissues of the skeleton.

Phenotypic Effects of Biglycan Deficiency Are Linked to Collagen Fibril Abnormalities, Are Synergized by Decorin Deficiency, and Mimic Ehlers-Danlos-Like Changes in Bone and Other Connective Tissues†

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. Ultrastructural analysis of bone collagen fibrils in bone of double‐KO mice reveals a complete loss of the basic fibril geometry with the emergence of marked “serrated fibril” morphology. The phenotype of the double‐KO animal mimics directly the rare progeroid variant of human Ehlers‐Danlos syndrome (EDS), in which skin fragility, progeroid changes in the skin (reduced hypodermis), and osteopenia concur as a result of impaired glycosaminoglycan (GAG) linking to bgn and dcn core proteins. Our data show that changes in collagen fibril morphology reminiscent of those occurring in the varied spectrum of human EDS are induced by both bgn deficiency and dcn deficiency in mice. The effects of an individual SLRP deficiency are tissue specific, and the expression of a gross phenotype depends on multiple variables including level of expression of individual SLRPs in different tissues and synergisms between different SLRPs (and likely other macromolecules) in determining matrix structure and functional properties.

Repair of craniotomy defects using bone marrow stromal cells.

BACKGROUND Techniques used to repair craniofacial skeletal defects parallel the accepted surgical therapies for bone loss elsewhere in the skeleton and include the use of autogenous bone and alloplastic materials. Transplantation of a bone marrow stromal cell population that contains osteogenic progenitor cells may be an additional modality for the generation of new bone. METHODS Full thickness osseous defects (5 mm) were prepared in the cranium of immunocompromised mice and were treated with gelatin sponges containing murine alloplastic bone marrow stromal cells derived from transgenic mice carrying a type I collagen-chloramphenicol acetyltransferase reporter gene to follow the fate of the transplanted cells. Control surgical sites were treated with spleen stromal cells or gelatin sponges alone, or were left untreated. The surgical defects were analyzed histologically for percent closure of the defect at 2, 3, 4, 6, and 12 weeks. RESULTS Cultured bone marrow stromal cells transplanted within gelatin sponges resulted in osteogenesis that repaired greater than 99.0+/-2.20% of the original surgical defect within 2 weeks. In contrast, cranial defects treated with splenic fibroblasts, vehicle alone, or sham-operated controls resulted in minimal repair that was limited to the surgical margins. Bone marrow stromal cells carrying the collagen transgene were immunodetected only in the newly formed bone and thus confirmed the donor origin of the transplanted cells. CONCLUSIONS These studies demonstrate that mitotically expanded bone marrow cells can serve as an abundant source of osteoprogenitor cells that are capable of repairing craniofacial skeletal defects in mice without the addition of growth or morphogenetic factors.

Mutations of the GNAS1 Gene, Stromal Cell Dysfunction, and Osteomalacic Changes in Non–McCune–Albright Fibrous Dysplasia of Bone

Activating missense mutations of the GNAS1 gene, encoding the α subunit of the stimulatory G protein (Gs), have been identified in patients with the McCune–Albright syndrome (MAS; characterized by polyostotic fibrous dysplasia, café au lait skin pigmentation, and endocrine disorders). Because fibrous dysplasia (FD) of bone also commonly occurs outside of the context of typical MAS, we asked whether the same mutations could be identified routinely in non‐MAS FD lesions. We analyzed a series of 8 randomly obtained, consecutive cases of non‐MAS FD and identified R201 mutations in the GNAS1 gene in all of them by sequencing cDNA generated by amplification of genomic DNA using a standard primer set and by using a novel, highly sensitive method that uses a protein nucleic acid (PNA) primer to block amplification of the normal allele. Histologic findings were not distinguishable from those observed in MAS‐related FD and included subtle changes in cell shape and collagen texture putatively ascribed to excess endogenous cyclic adenosine monophosphate (cAMP). Osteomalacic changes (unmineralized osteoid) were prominent in lesional FD bone. In an in vivo transplantation assay, stromal cells isolated from FD failed to recapitulate a normal ossicle; instead, they generated a miniature replica of fibrous dysplasia. These data provide evidence that occurrence of GNAS1 mutations, previously noted in individual cases of FD, is a common and perhaps constant finding in non‐MAS FD. These findings support the view that FD, MAS, and nonskeletal isolated endocrine lesions associated with GNAS1 mutations represent a spectrum of phenotypic expressions (likely reflecting different patterns of somatic mosaicism) of the same basic disorder. We conclude that mechanisms underlying the development of the FD lesions, and hopefully mechanism‐targeted therapeutic approaches to be developed, must also be the same in MAS and non‐MAS FD.

In vivo bone formation by human bone marrow stromal cells: Effect of carrier particle size and shape

Successful closure of bone defects in patients remains an active area of basic and clinical research. A novel and promising approach is the transplantation of human bone marrow stromal cells (BMSCs), which have been shown to possess a significant osteogenic potential. The extent and quality of bone formation by transplanted human BMSCs strongly depends on the carrier matrix with which cells are transplanted; to date, hydroxyapatite/tricalcium phosphate (HA/TCP) supports far more osteogenesis than any other matrix tested. In order to further improve the technique of BMSC transplantation, we studied whether commercially available HA/TCP particles, clinically approved as an osteoconductive material and commercially available as particles measuring 0.5-1.0 mm diameter, is an optimum matrix for promoting bone development by BMSCs. HA/TCP and HA particles of varying size were sieved into a variety of size ranges, from <0.044 mm to 1.0-2.0 mm. Transplants were formed by mixing 40 mg aliquots of particles with cultured passaged human BMSCs. They were placed in subcutaneous pockets in immunocompromised Bg-Nu-XID mice and harvested 4 or 10 weeks later. The transplants were examined histologically; the presence of bone within each transplant was evaluated using histomorphometry or blindly scored on a semiquantitative scale. Transplant morphology and the amount of new bone varied in a consistent fashion based on particle size and shape. Transplants incorporating HA/TCP particles of 0.1-0.25 mm size demonstrated the greatest bone formation at both 4 and 10 weeks; larger or smaller particles were associated with less extensive bone formation, while a size of 0.044 mm represented a threshold below which no bone formation could be observed. Flat-sided HA particles measuring 0.1-0.25 mm formed no bone. The differences in bone formation were not attributable to the differences in cell attachment among the groups. Instead, the size and spatial and structural organization of the particles within BMSC transplants appear to determine the extent of bone formation. These findings provide necessary information for the successful clinical application of BMSC transplantation techniques.

EFFECT OF SERUM ON HUMAN BONE MARROW STROMAL CELLS: EX VIVO EXPANSION AND IN VIVO BONE FORMATION

Background. Bone marrow stromal cell (BMSC) transplantation may offer an efficacious method for the repair of bone defects.This approach has been developed using BMSCs expanded ex vivo in medium with fetal bovine serum (FBS). For clinical applications, however, contact of BMSCs with FBS should be minimized. We studied the effect of FBS substitutes on both human BMSC proliferation in vitro and subsequent bone formation in vivo. Methods. BMSC proliferation was measured by colony forming efficiency (CFE) and by cell numbers at consecutive passages. Bone formation was studied in 6- to 8-week-old transplants of human BMSCs in immunocompromised mice. Results. Medium with FBS was more effective in stimulating BMSC proliferation than medium with either human serum (HS) or rabbit serum (RS). Compared to bone formed by BMSCs cultured continuously with FBS, bone formed by cells cultured with HS, or with FBS switched to HS, was considerably less extensive, while bone formed by cells cultured with FBS switched to serum-free medium (SFM) was considerably more extensive. The increase in bone formation was due to neither the SFM components nor to the proliferation status of BMSCs prior to transplantation. Conclusions. Our data demonstrate that for ex vivo expansion of human BMSCs, medium with FBS remains most effective. However, incubation of human BMSCs in SFM prior to in vivo transplantation significantly stimulates subsequent bone formation. This finding increases the practicality of using culture-expanded BMSCs for autologous human transplantation and suggests the presence of osteogenic inhibitors in serum.

Normal Human Cementum‐Derived Cells: Isolation, Clonal Expansion, and In Vitro and In Vivo Characterization

Cultures of primary human cementum‐derived cells (HCDCs) were established from healthy premolar teeth extracted for orthodontic reasons. Cementum was manually dissected, fragmented, and digested twice with collagenase. Following a thorough wash to remove liberated cells, the remaining cementum fragments were plated in Dulbeccos modified Eagles medium/F12 medium containing 10% fetal bovine serum. Discrete colonies that contained cells exhibiting fibroblast‐like morphology were visible after 14–21 days of culture. When the colonies became sufficiently large, cells from individual colonies were isolated and subcultured. Cementum‐derived cells exhibited low levels or no alkaline phosphatase activity and mineralized in vitro to a lesser degree than human periodontal ligament (PDL) cells and human bone marrow stromal cell (BMSC) cultures. To study differentiation capacities of HCDCs, cells were attached to hydroxyapatite/tricalcium phosphate ceramic and transplanted subcutaneously into immunodeficient mice. The transplants were harvested 3, 6, and 8 weeks after transplantation and evaluated histologically. In human BMSC transplants, new bone tissue was formed with a prominent osteoblastic layer and osteocytes embedded in mineralized bone matrix. No osseous tissue was formed by PDL cells. Of six single colony‐derived strains of HCDCs tested, three formed a bone‐like tissue that featured osteocyte/cementocyte‐like cells embedded within a mineralized matrix and which was lined with a layer of cells, although they were somewhat more elongated than osteoblasts. These results show that cells from normal human cementum can be isolated and expanded in vitro. Furthermore, these cells are capable of differentiating and forming mineralized tissue when transplanted into immunodeficient mice.

Circulating Connective Tissue Precursors: Extreme Rarity in Humans and Chondrogenic Potential in Guinea Pigs

Using a variety of cell separation techniques and cultivation conditions, circulating, adherent, connective tissue, clonogenic cells were found in just 3 donors out of 66, demonstrating that these precursors are extremely rare in postnatal human blood. Contrary to humans, guinea pig blood shows much more reproducible connective tissue colony formation; it was therefore chosen to study the differentiation potential of adherent blood‐derived clonogenic cells. Out of 22 single colony‐derived strains of various morphologies, only 5 spindle‐shaped strains showed extensive proliferative capacity in vitro. None of these strains formed bone upon in vivo transplantation, whereas two strains formed cartilage in high‐density pellet cultures in vitro. Both chondrogenic strains included cells expressing aggrecan, whereas nonchondrogenic strains did not. Out of four polyclonal strains studied, one formed both cartilage and abundant bone accompanied by hematopoiesis‐supporting stroma. Evidently, there are cells in adult guinea pig blood capable of both extensive proliferation and differentiation toward cartilage: circulating chondrogenic precursors. Although some of these cells lack osteogenic potential and therefore represent committed chondrogenic precursors, others may be multipotential and consequently belong to the family of skeletal stem cells. This is the first demonstration of postnatal circulating chondrogenic precursors, as well as of precursor cells with chondrogenic but not osteogenic potential.

Enumeration of the colony-forming units-fibroblast from mouse and human bone marrow in normal and pathological conditions

Bone marrow stromal cell populations, containing a subset of multipotential skeletal stem cells, are increasingly contemplated for use in tissue engineering and stem cell therapy, whereas their involvement in the pathogenetic mechanisms of skeletal disorders is far less recognized. We compared the concentrations of stromal clonogenic cells, colony forming units-fibroblast (CFU-Fs), in norm and pathology. Initially, culture conditions were optimized by demonstrating that fetal bovine serum heat inactivation could significantly repress colony formation. Using non-heat-inactivated fetal bovine serum, the concentration of CFU-Fs (colony-forming efficiency, CFE) ranged from 3.5 +/- 1.0 to 11.5 +/- 4.0 per 1 x 10(5) nucleated cells in five inbred mouse strains. In four transgenic lines with profound bone involvement, CFE was either significantly reduced or increased compared to wild-type littermates. In normal human donors, CFE decreased slightly with age and averaged 52.2 +/- 4.1 for children and 32.3 +/- 3.0 for adults. CFE was significantly altered in patients with several skeletal, metabolic, and hematological disorders: reduced in congenital generalized lipodystrophy, achondroplasia (SADDAN), pseudoachondroplasia, and Paget disease of bone and elevated in alcaptonuria and sickle cell anemia. Our findings indicate that under appropriate culture conditions, CFE values may provide useful insights into bone/bone marrow pathophysiology.

Cementum‐Forming Cells Are Phenotypically Distinct from Bone‐Forming Cells

Normal human cementum‐derived cells (HCDCs), expanded in vitro, formed mineralized matrix when attached to a ceramic carrier and transplanted subcutaneously into immunodeficient mice. The mineralized matrix elaborated by transplanted HCDC exhibited several features identical to cementum in situ and was significantly different from bone deposited by similarly transplanted human bone marrow stromal cells (BMSCs). No bone marrow formation and very few or no tartrate‐resistant acid phosphatase (TRAP)‐positive cells (osteoclasts and osteoclastic precursors) were found in HCDC transplants. In contrast, in BMSC transplants both hematopoiesis and TRAP‐positive cells were routinely observed. Furthermore, compared with BMSC‐derived matrix, HCDC‐derived matrix was less cellular, numerous empty lacunae were present, and fewer cells were found on the cementum matrix/ceramic carrier interface. The organization of collagen fibers in HCDC‐derived matrix, as visualized by using the Picrosirus red staining method, was similar to cementum, with typical unorganized bundles of collagen fibers. In contrast, bone matrix elaborated by transplanted BMSC had lamellar structure, identical to mature bone in situ. Finally, cementocytes embedded in the cementum‐like matrix were immunopositive for fibromodulin and lumican, whereas osteocytes within the bonelike matrix were negative. This pattern is consistent with the cementum and bone in situ, respectively. These results indicate that human cementum cells are phenotypically distinct from bone cells and provide further validation of the combined in vitro/in vivo model of human cementogenesis recently developed in our laboratory.