Structural analysis of the extension peptides on matrix forms of type V collagen in fetal calf bone and skin.

The structure of the extension peptides retained on the tissue form of type V collagen molecules was determined. Type V collagen alpha chains containing extension peptides were extracted from fetal calf skin and bone by 4 M guanidine-HCl and 0.5 M acetic acid, respectively. Collagens present in both extracts were fractionated by sodium chloride precipitation. The collagen alpha(V) chains were then resolved by reverse-phase high performance liquid chromatography. The N-terminal extension peptides were characterized by direct sequence analysis after deblocking with pyroglutamate amino-peptidase and analysis of the products of digestion by bacterial collagenase, chymotrypsin, V8 protease and endoproteinase Lys-C. The results showed that the retained extension peptides on type V collagen molecules in the extracellular matrix of skin and bone were amino-propeptides and that the alpha 2(V) chain retains an intact amino-propeptide while the alpha 1(V) chain appears to be partially processed. The extended alpha 1(V) chain isolated from fetal calf bone gave an identical amino-terminal sequence to that of the alpha 1(V) chain isolated from fetal calf skin, suggesting that a specific enzyme may be involved in processing the alpha 1(V) amino-propeptide.

The carboxypropeptide trimer of type II collagen is a prominent component of immature cartilages and intervertebral-disc tissue.

Immature bovine cartilages and intervertebral-disc tissue all revealed a prominent protein, not present in the adult tissues, in non-denaturing extracts made with chondroitin ABC lyase (EC 4.2.2.4), Streptomyces hyaluronidase (EC 4.2.2.1) or 1 M NaCl. The protein ran on SDS-polyacrylamide electrophoresis, before disulphide reduction, as a close doublet of bands of apparent molecular weight 110,000 and 105,000. After reduction, they dissociated respectively into two protein bands at 37,000 and 35,000, indicating that the initial molecules were disulphide-bonded trimers. Amino-terminal sequence analysis established the identity of both proteins (Mr 110,000 and Mr 105,000) as forms of the carboxypropeptide of type II collagen. The larger molecule appeared to be the trimer of intact alpha 1(II) carboxypropeptides and the smaller, a version composed of chains that were ten residues shorter at their amino-terminal ends. The material appears to be identical to chondrocalcin, a protein previously found to be enriched in fetal growth plate and named on the basis that it may play a role in cartilage calcification. The present findings, however, indicate that the protein is equally abundant in all type II collagen-synthesizing young cartilages, including nucleus pulposus of the intervertebral disc and other cartilages that never calcify.

Collagens in an adult bovine medial collateral ligament: immunofluorescence localization by confocal microscopy reveals that type XIV collagen predominates at the ligament-bone junction.

To understand the structure and function of medial collateral ligament, collagens present in an adult bovine ligament were determined. The mid-section of the ligament was powdered and extracted with 4M guanidinium hydrochloride, and the residue was digested with pepsin to solubilize the collagens. Type I collagen was the major fibril collagen recovered in the pepsin solubilized fraction, with types III and V each representing about 5% and 2%, respectively. Type VI collagen was the major collagen present in the guanidinium hydrochloride extract, and it accounted for about 40% of the proteins in the extract or 4% of the tissue dry weight. Type XII and XIV collagens were also detected in the guanadinium hydrochloride extract as minor components. Immunofluorescence localization using confocal microscopy showed that type XII and XIV collagens are associated with the ligament fibrillar network and that type XIV collagen was prominent at the ligament-bone junction. These data reinforce the notion that these collagens are associated with the type I collagen fibrillar network in connective tissues. In view of high mechanical stresses that exist at the ligament-bone interface, presence of type XIV collagen in high concentration at this junction may contribute to the modulation of the biomechanical properties of this tissue.

Identification of types II, IX and X collagens at the insertion site of the bovine achilles tendon.

Achilles tendinous collagen fibrils insert into the calcaneus by first passing through a zone that is defined histologically as fibrocartilaginous. This zone consists of four regions: tendon proper, non-mineralized and mineralized fibrocartilage and bone. The function of this zone has not yet been clearly defined. To gain more insight into the role of this fibrocartilaginous zone, collagens present in the zone of the Achilles tendon-calcaneus interface were isolated and characterized. Types II, IX and X collagens were identified in the pepsin digests of the tissue harvested from the bovine Achilles tendon-calcaneus interface. Western blotting using specific antisera to types II, IX and X collagens confirmed the identity of these collagens. Immunofluorescence localization placed type X collagen predominantly in the mineralized zone of the tendon-calcaneus junction, while type IX collagen was distributed throughout the the insertion site. The presence of the cartilage-specific collagens at the Achilles tendon-calcaneus-interface suggests that this zone is cartilaginous in nature. The presence of type X collagen at this junction is not clear, but our present findings go along with the previous report which showed that type X collagen is present in the mineralized zone of the medial collateral ligament femoral insertion site. These data suggest that type X collagen may be a resident of mineralized fibrocartilaginous zones of tendon or ligament-bone junctions and may participate in anchoring ligament or tendon to bone.

Identification of the cartilage alpha 1(XI) chain in type V collagen from bovine bone.

Type V collagen prepared from bovine bone was resolved into three distinct alpha-chains by high performance liquid chromatography and gel electrophoresis. Peptide mapping established two chains as alpha 1(V) and alpha 2(V) as expected and the third as the cartilage alpha 1(XI) chain (previously thought to be unique to cartilage). In adult bone, the type V collagen fraction was richer in alpha 1(XI) chains than in fetal bone (about 1/3 of the chains in the adult). How these polypeptides are organized into native molecules is not yet clear, though the stoichiometry suggests cross-type heterotrimers between the type V and XI chains.

The effects of age on rabbit MCL fibroblast matrix synthesis in response to TGF-beta 1 or EGF.

In this study, we examined the effects of age on collagen and total protein synthesis by ligament fibroblasts in response to growth factors. Three different doses of transforming growth factor-beta 1 (TGF-beta 1) or epidermal growth factor (EGF) were individually added to in vitro fibroblast cultures from the medial collateral ligament (MCL) of skeletally immature (age 3 months), mature (age 12 months) and senescent (age 48-51 months) rabbits. Analysis of the effects of age revealed that fibroblasts from senescent rabbits produced significantly less collagen in response to TGF-beta 1 or EGF stimulation when compared to fibroblasts from immature rabbits. Furthermore, increased age was found to result in significant reductions in the baseline levels of collagen synthesis but not total protein synthesis. Additionally, collagen and total protein synthesis by MCL fibroblasts were significantly affected by the TFG-beta 1 dose, but not by the EGF dose. When fibroblasts were normalized to their own controls, the increase in collagen and total protein synthesis due to TGF-beta 1 and EGF for the senescent group were found to be greater than those for the skeletally immature rabbits at all doses. This demonstrates that MCL fibroblasts from senescent rabbits are responsive to growth factors.

Effect of rhBMP-2 on the osteogenic potential of bone marrow stromal cells from an osteogenesis imperfecta mouse (oim).

To understand whether osteogenesis imperfecta (OI) could result from defective differentiation of osteoprogenitor cells, we investigated the osteogenic potential of bone marrow stromal cells from a mouse model of human OI (oim). Bone marrow was flushed from the femurs and tibias of oim and normal littermates using a syringe with Dulbecco's modified Eagle's medium, and cells were allowed to adhere to flasks. Adherent cells were trypsinized and passaged weekly at a 1:4 split. The established stromal cells were assessed for collagen synthesis, alkaline phosphatase, and osteocalcin production in the presence or absence of rhBMP-2. The stromal cells were also assessed for mineralization by Von-Kossa staining and for exogenous gene transfer using adeno-lacZ and a retroviral vector. The bone marrow stromal cells from oim mice synthesized alpha 1(I) homotrimers as expected, whereas the stromal cells from the normal littermates synthesized alpha 1(I)2 alpha 2(I) heterotrimers. The bone marrow stromal cells exhibited low levels of alkaline phosphatase activity under basal conditions: upon treatment with rhBMP-2, the level of the alkaline phosphatase activity increased approximately 40-fold. Cytochemical staining of the cells confirmed the expression of alkaline phosphatase by the oim stromal cells and its augmentation by rhBMP-2. Osteocalcin production in the stromal cells was also enhanced approximately threefold by rhBMP-2. oim stromal cells grown in the presence of beta-glycerophosphate and ascorbic acid demonstrated Von-Kossa-positive solid deposits after 3 weeks in culture. Ten days after infection with adeno-lacZ, approximately 70% of oim stromal cells expressed the transgene product, and after infection with a retrovirus, approximately 20% of the cells expressed the transgene. These data indicate that bone marrow stromal cells, have osteogenic potential, and also the potential to be transduced with exogenous genes. Under basal conditions, however, the stromal cells from oim mice exhibited significantly lower levels of alkaline phosphatase activity than their normal littermates.

Novel approaches to fracture healing.

The fractures that occur as a result of trauma frequently require multiple stage surgical procedures to achieve adequate union. Bone grafting with autogenous cancellous or cortico cancellous bone grafts is the traditional method used to repair bone defects. Most fractures will heal using this traditional procedure, however a number of fractures, up to 10% of the cases in United States alone, will result in delayed or impaired healing. Novel approaches are currently being investigated for the augmentation and acceleration of fracture healing. Some of these approaches include the use of biodegradable matrices; cell based approaches supplemented with osteogenic factors and genetic therapy. Cell based approaches for fracture healing have roused intense interest because of the great advance in the isolation and expansion of cells from the marrow that have the ability to differentiate into various types of cells including osteoblasts. In addition, the discovery and cloning of several proteins (bone morphogenetic proteins) that have the ability to induce bone formation, have contributed to the investigation of novel approaches to augment fracture healing. Use of genetic therapy for the augmentation of fracture healing has also recently gained strong interest. The attractive feature of gene therapy is that therapeutic proteins can be delivered locally to the fracture site in relatively high concentrations and in a sustained fashion. This review discusses these novel approaches and presents an assessment of their future clinical applicability.

In vivo expression of human growth hormone by genetically modified murine bone marrow stromal cells and its effect on the cells in vitro.

Human growth hormone (hGH) is frequently used clinically for growth abnormalities in children and also in adults with growth hormone deficiency. The hormone is usually administered to the individuals by frequent injections. In the present study we investigated the potential of bone marrow stromal cells as vehicles to deliver the GH in vivo by infusion of cells transduced with hGH cDNA into mice femurs. The effect of the hormone on the transduced cells in vitro was also assessed. Bone marrow stromal cells established from a mouse model of human osteogenesis imperfecta mice (oim) were transduced with a retrovirus containing hGH and neomycin resistance genes. The hGH-expressing cells were selected in a medium containing G418 and were then assessed for the hGH expression in vitro. The selected cells synthesized 15 ng/10(6) cells of hGH per 24 h in vitro and exhibited alkaline phosphatase activity when they were treated with the human recombinant bone morphogenetic protein 2 (rhBMP-2). The transduced cells also proliferated faster than the LacZ transduced cells but they did not exhibit a higher rate of matrix synthesis. When 2 x 10(6) hGH+ cells were injected into the femurs of mice, hGH was detected in the serum of the recipient mice up to 10 days after injection. The highest level of growth hormone expression, 750 pg/ml, was detected in the serum of the recipient mice I day after injection of the transduced cells. hGH was also detected in the medium conditioned by cells that were flushed from the femurs of the recipient mice at 1, 3, and 6 days after cell injection. These data indicate that bone marrow stromal cells could potentially be used therapeutically for the delivery of GH or any other therapeutic proteins targeted for bone. The data also suggest that GH may exert its effects on bone marrow stromal cells by increasing their rate of proliferation.

Increased matrix synthesis following adenoviral transfer of a transforming growth factor beta1 gene into articular chondrocytes.

Monolayer cultures of lapine articular chondrocytes were transduced with first-generation adenoviral vectors carrying lacZ or transforming growth factor beta1 genes under the transcriptional control of the human cytomegalovirus early promoter. High concentrations of transforming growth factor beta1 were produced by chondrocytes following transfer of the transforming growth factor beta1 gene but not the lacZ gene. Transduced chondrocytes responded to the elevated endogenous production of transforming growth factor beta1 by increasing their synthesis of proteoglycan, collagen, and noncollagenous proteins in a dose-dependent fashion. The increases in collagen synthesis were not accompanied by alterations in the collagen phenotype; type-II collagen remained the predominant collagen. Transforming growth factor beta1 could not, however, rescue the collagen phenotype of cells that had undergone phenotypic modulation as a result of serial passaging. These data demonstrate that chondrocytes can be genetically manipulated to produce and respond to the potentially therapeutic cytokine transforming growth factor beta1. This technology has a number of experimental and therapeutic applications, including those related to the study and treatment of arthritis and cartilage repair.

Medial collateral ligament healing one year after a concurrent medial collateral ligament and anterior cruciate ligament injury: an interdisciplinary study in rabbits.

The optimal treatment for concurrent injuries to the medial collateral and anterior cruciate ligaments has not been determined, despite numerous clinical and laboratory studies. The objective of this study was to examine the effect of surgical repair of the medial collateral ligament on its biomechanical and biochemical properties 52 weeks after such injuries. In the left knee of 12 skeletally mature New Zealand White rabbits, the medial collateral ligament was torn and the anterior cruciate ligament was transected and then reconstructed. This is an experimental model previously developed in our laboratory. In six rabbits, the torn ends of the medial collateral ligament were repaired, and in the remaining six rabbits, the ligament was not repaired. Fifty-two weeks after injury, we examined varus-valgus and anterior-posterior knee stability; structural properties of the femur-medial collateral ligament-tibia complex; and mechanical properties, collagen content, and mature collagen crosslinking of the medial collateral ligament. We could not detect significant differences between repair and nonrepair groups for any biomechanical or biochemical property. Our data support clinical findings that when the medial collateral and anterior cruciate ligaments are injured concurrently and the anterior cruciate ligament is reconstructed, conservative treatment of the ruptured medial collateral ligament can result in successful healing.

Effect of growth factors on matrix synthesis by ligament fibroblasts.

Although it has been reported that several growth factors modulate soft-tissue healing, the specific effects of growth factors on protein synthesis during ligament healing have not been widely investigated. In this study, we examined the effects of basic and acidic fibroblast growth factors, transforming growth factor beta 1, and epidermal growth factor on collagen and noncollagenous protein synthesis by cultured fibroblasts from medial collateral ligament and anterior cruciate ligament in vitro. Uptake of tritiated proline was used to measure synthesis of collagen and noncollagenous protein, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to analyze the type of collagens synthesized. Our data showed that transforming growth factor beta 1 increased both collagen and noncollagenous protein synthesis by medial collateral and anterior cruciate ligament fibroblasts on a dose-dependent basis. Collagen synthesis by cultured fibroblasts from the medial collateral and anterior cruciate ligaments was increased by treatment with transforming growth factor beta 1 by as much as approximately 1.5 times that of untreated controls. Although the response to transforming growth factor beta 1 by anterior cruciate ligament fibroblasts was equal to that by medial collateral ligament fibroblasts, the amounts of matrix proteins synthesized by anterior cruciate ligament fibroblasts were approximately half of that by medial collateral ligament fibroblasts. The increase was mostly in type-I collagen. Treatment of anterior cruciate ligament fibroblasts with epidermal growth factor increased collagen synthesis by approximately 25% but had little effect on medial collateral ligament fibroblasts. Neither basic nor acidic fibroblast growth factor increased either collagen or noncollagenous protein synthesis. These findings suggest that topical application of transforming growth factor beta 1, alone or in combination with epidermal growth factor, may have the potential to strengthen the ligament by increasing matrix synthesis during its remodeling and healing processes.

Cytokine-induced tendinitis: a preliminary study in rabbits.

This study was designed to determine the effects of a single injection of a species-specific preparation of cytokines into rabbit patellar tendons and to compare the results with a known model of tendinitis, the collagenase-injection model. New Zealand White rabbits were divided into two groups and two time periods (4 and 16 weeks) and injected in the midsubstance of the right patellar tendon with either cytokines or collagenase under ultrasound guidance to confirm intratendinous needle placement. The left patellar tendon was injected with 0.025 ml of saline solution and served as a control. The rabbits were returned to cage activity after injection. At death, two rabbits in each group underwent histological analysis; the remaining eight animals in each time frame were evaluated biomechanically and then biochemically with use of the patella/whole patellar tendon/tibia complex. Histologic results at 4 weeks in the tendons injected with cytokines demonstrated increased cellularity, which was resolving by 16 weeks. The matrix appeared unchanged. The tendons injected with collagenase demonstrated increased angiogenesis of the matrix, hypercellularity, and fibrosis around the tendon at 4 weeks. At 16 weeks, myxoid changes, focal fibrosis, and collagen-bundle disarray with persistent increase in cellularity were noted. Biomechanically, a significant decrease in ultimate load at 16 weeks was seen in the tendons injected with cytokines but no change was seen in cross-sectional area. The tendons injected with collagenase demonstrated a significant increase in cross-sectional area at 4 and 16 weeks compared with those injected with cytokines. Biochemically, there was no significant difference in collagen content between the two groups at 4 or 16 weeks but the tendons injected with collagenase demonstrated a significant increase in crosslinking at 16 weeks. Our conclusion is that the tendons injected with the cytokine preparation represent a model of mild, seemingly reversible tendon injury. The cytokine preparation produces no matrix damage or evidence of collagen degradation and is species specific.

Transfer of lacZ marker gene to the meniscus.

BACKGROUND: Lesions in the avascular two-thirds of the meniscus do not heal well and are of concern clinically. Various growth factors promote the synthesis of matrix by meniscal cells and thus have the potential to augment healing. However, their clinical application is severely hindered by problems with delivery. An attractive approach to overcoming such problems is to transfer genes that encode the growth factors in question to the site of the injury. As a prelude to this, we evaluated methods for delivering genes to the meniscus. METHODS: Gene transfer was evaluated in vitro and in vivo with a lacZ marker gene, which expresses the enzyme beta-galactosidase. Two types of vectors were tested: an adenovirus and a retrovirus. Monolayers of lapine, canine, and human meniscal cells, as well as intact lapine and human menisci, were used for the in vitro studies. Lesions were created in the menisci of rabbits and dogs for the in vivo studies. Gene transfer to the sites of the experimental meniscal lesions in vivo was accomplished in two ways. In the lapine model, a suspension of adenovirus carrying the lacZ marker gene was mixed with whole blood and the clot was inserted into the lesion. In the canine model, retrovirally transduced allogenic meniscal cells carrying the lacZ marker gene were embedded in collagen gels and transferred to the defects. The animals were killed at various time-points, and gene expression was evaluated by histological examination of sections stained with 5-bromo-4-chloro-indolyl-beta-D-galactose (X-gal), from which a blue chromagen is released in the presence of beta-galactosidase. RESULTS: Monolayer cultures of lapine, canine, and human meniscal cells were susceptible to genetic transduction by both adenoviral and retroviral vectors. In vitro gene transfer to intact human and lapine menisci proved possible both by direct, adenoviral, delivery and indirect, retroviral, delivery. Gene expression persisted for at least twenty weeks under in vitro conditions. With regard to the in vivo studies, gene expression persisted within the clot and in some of the adjacent meniscal cells for at least three weeks in the lapine defect model. In the canine defect model, gene expression persisted within the transplanted, transduced meniscal cells for at least six weeks. CONCLUSIONS: It is possible to transfer genes to sites of meniscal damage and to express them locally within the lesion for several weeks.

Interaction of TGF-beta1 and rhBMP-2 on human bone marrow stromal cells cultured in collagen gel matrix.

Transforming growth factor-beta1 (TGF-beta1) and bone morphogenetic protein-2 (BMP-2) are abundant proteins in the bone matrix. However, their interaction in controlling osteoblast differentiation is not clearly understood. In this study, HBMSCs were cultured in collagen gel matrix with different condition of exogenous rhBMP-2 and TGF-beta1 in order to determine the interaction of BMP-2 and TGF-beta1 on human bone marrow stromal cells (HBMSCs) differentiation. The cultured cells were analyzed for cell proliferation, alkaline phophatase (ALP) activity and mineralization staining with Von-Kossa. The cells treated with TGF-beta1 exhibited a higher rate of cell growth than those without. However, the cells cultured in collagen gel matrix showed a lower rate of cell growth than the cells cultured in a monolayer. To investigate the effects of both cytokines on osteoblast differentiation, the cells were treated with 0, 1, 5, 10 ng/ml of TGF-beta1 for 2 days. This was followed by culturing with 0, 1, 5, and 10 ng/ml of TGF-beta1 and 100 ng/ml of rhBMP-2 together for 3 days with the alkaline phosphatase (ALP) activity measured. The cells treated with 1 ng/ml of TGF-beta1 responded efficiently to rhBMP-2 and expressed ALP activity with a level equivalent to that exhibited by cells that were not treated with TGF-beta1. The cells treated with 5 and 10 ng/ml of TGF-beta1 showed a dramatic decrease in ALP activity. The cells treated with 10 ng/ml of TGF-beta1 followed by rhBMP-2 alone exhibited an intermediate ALP activity. The cells treated with 100 ng/ml of rhBMP-2 demonstrated Von-Kossa positive solid deposits after 3 weeks, while there were few Von-Kossa positive solid deposits when the cells treated with 10 ng/ml of TGF-beta1. These results show that TGF-beta1 inhibits the effects of rhBMP-2 on the osteoblast differentiation of HBMSCs in a dose dependant manner. Furthermore, the effects of TGF-beta1 on HBMSCs are reversible. This suggest that TGF-beta1 and rhBMP-2 are coordinately controlled during the osteoblast differentiation of HMBSCs.

Structural characteristics of cross-linking sites in type V collagen of bone. Chain specificities and heterotypic links to type I collagen.

To understand the role of type V collagen and its spatial interrelationship with type I collagen in bone matrix, the molecule's covalent intermolecular cross-links were structurally characterized. Type V collagen containing alpha 1(V), alpha 2(V) and alpha 1(XI) chains was isolated from bovine bone and reacted with NaB3H4 to label the cross-linking residues. Radiolabeled native molecules and isolated alpha chains were treated with sodium metaperiodate to cleave the divalent cross-linking bonds. Sequence analysis of the periodate-released peptides matched two of them to alpha 1(V) and alpha 1(XI) aminopropeptide domains. A third peptide was derived from the alpha 1(I) carboxytelopeptide domain of type I collagen. This latter peptide, therefore, came from a site of heterotypic cross-linking between types I and V collagens and accounted for about 15% of the total cross-linked peptides. Sequence analysis of isolated cross-linked tryptic peptides defined the helical sites of attachment of the periodate-released telopeptides and revealed that the putative aminoproteinase-cleavage sites in the alpha 1(V) and alpha 1(XI) chains are located in the molecule interior to the cross-linking residue. These data imply that type V collagen molecules in the extracellular matrix are primarily cross-linked to each other in a head-to-tail linear polymer that is linked laterally to type I collagen molecules in copolymeric fibrils.

Bone type V collagen: chain composition and location of a trypsin cleavage site.

The component alpha-chains of type V collagen from bovine bone were isolated and structurally characterized by gel electrophoresis, high performance liquid chromatography (HPLC) and amino acid sequence analysis. Three distinct alpha-chains were identified. Two of these were the well described alpha 1 (V) and alpha 2 (V) chains; the third proved to be identical to the cartilage alpha 1 (XI) chain. In adult bone the ratio between the three chains was about 1:1:1. Native type V collagen was cleaved by trypsin at 33 degrees C or 37 degrees C into 3/5 fragments. Aminoterminal sequence analysis of the alpha 1 (V) and alpha 1 (XI) fragments showed they both resulted from trypsin cleavage between residue 434 and 435. Trypsin apparently cleaves the type V molecule within a relatively unstable domain of the triple helix which presumably may also be a natural site of initial cleavage by a protease in vivo.

Identification and immunolocalization of type X collagen at the ligament-bone interface.

In some ligaments, ligamentous collagen fibrils attach to bone by first passing through non-mineralized and mineralized fibrocartilage present at the ligament-bone interface. To understand better the function of these fibrocartilages, collagens present at the femoral insertion of the bovine medial collateral ligament were isolated and characterized. Types II and IX collagens were identified in pepsin digests of the tissue in addition to type X collagen originally thought to be associated with the cartilages undergoing endochondral bone formation. Presence of type X collagen was confirmed by immunoblotting and by immunofluorescence localization using laser confocal microscopy. Type X collagen was localized predominantly in the mineralized zone of the ligament insertion. These data indicated that type X collagen may play a role in ligament attachment to bone.

A 92 kDa gelatinase (MMP-9) cleavage site in native type V collagen.

Native type V collagen molecules resist mammalian collagenase but are cleaved by certain gelatinases. We report a prominent site of cleavage within the collagen type V molecules by 92 kDa gelatinase (MMP-9). The enzyme was purified from conditioned medium of a rabbit synovial cell line (HIG-82). It cleaved native type V collagen from bovine bone in solution at two molecular sites, one near the amino-terminus, the other producing a 3/5 C-terminal fragment. Amino-terminal sequence analysis of the individual alpha chains from this latter fragment showed that MMP-9 had cleaved between residues Gly439-Val in both alpha 1(V) and alpha (XI) and between residues Gly445-Leu in the alpha 2(V) chain. These sites are close to the previously reported trypsin-cleavage site. The findings imply that gelatinases may be necessary for initiating or completing degradation of type I/type V copolymeric fibrils for growth and remodeling of extracellular collagen.

Gene therapy approaches for osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a heterogeneous group of genetic disorders that affect connective tissue integrity. The hallmark of OI is bone fragility, although other manifestations, which include osteoporosis, dentigenesis imperfecta, blue sclera, easy bruising, joint laxity and scoliosis, are also common among OI patients. The severity of OI ranges from prenatal death to mild osteopenia without limb deformity. Most forms of OI result from mutations in the genes that encode either the proalpha1or proalpha2 polypeptide chains that comprise type I collagen molecules, the major structural protein of bone. Treatment depends mainly on the severity of the disease with the primary goal to minimize fractures and maximize function. Current treatments include surgical intervention with intramedullarly stabilization and the use of prostheses. Pharmacological agents have also been attempted with limited success with the exception of recent use of bisphosphonates, which have been to shown to have some effect. Since OI is a genetic disease, these agents are not expected to alter the course of the collagen mutations. Cell and gene therapies as potential treatments for OI are therefore currently being actively investigated. The design of gene therapies for OI is however complicated by the genetic heterogeneity of the disease and by the factor that most of the OI mutations are dominant negative where the mutant allele product interferes with the function of the normal allele. The present review will discuss the molecular changes seen in OI, the current treatment options and the gene therapy approaches being investigated as potential future treatments for OI.