Marcel E. Nimni

Polypeptide growth factors: targeted delivery systems

Growth factors are becoming extremely valuable tools in our attempts to understand the mechanisms that modulate cellular activities. Their targeting to appropriate cells and maintaining adequate pharmacological levels becomes essential, particularly in view of the different effects that these compounds have on various cells and the dose dependence of their response. Within this context, this review focuses primarily on the delivery of growth factors involved in the processes of wound healing and tissue repair.

Collagen defect induced by penicillamine.

Collagen synthesis, as judged by the accumulation of collagen in a subcutaneous, induced granuloma, was significantly decreased by penicillamine. Penicillamine also caused a marked increase in the amount of soluble collagen in skin and a sharp drop in insoluble material. These findings, which reflect an abnormal pattern of collagen metdbolism, are accompanied by an inhibition of wound-healing and by skin fragility.

Modulation of Collagen Synthesis by Transforming Growth Factor-β in Keloid and Hypertrophic Scar Fibroblasts

Keloid and hypertrophic scars are fibrous growths characterized by overabundant collagen deposition. We examined the effect of transforming growth factor-β (TGF-β), a known stimulant for the production of connective tissue matrices, on the rate of collagen synthesis in keloid fibroblasts (KFs), hypertrophic scar fibroblasts (HSFs), and normal skin fibroblasts (NSFs). Fibroblasts were cultured in three-dimensional fibrin-gel matrices in the presence or absence of TGF-β (5 ng/ml) or anti–TGF-β neutralizing antibody (50 μ/ml). Secreted collagen levels, labeled with 3H-proline, were measured after 48 hours. KFs produced up to 12 times more collagen than NSFs, and up to 4 times more than HSFs. Although KFs increased their rate of collagen production by up to 2.7 times in response to TGF-β, HSFs and NSFs did not (p = 0.065). Anti–TGF-β antibody reduced the rate of collagen synthesis of KFs by 40% (p = 0.003), although it did not suppress collagen production in HSFs (p = 0.06) and NSFs (p = 0.75). We conclude that although KFs and HSFs are similar in that they both overproduce collagen, they are different in that only KFs display a marked sensitivity to TGF-β, which is abundant during the proliferative phase of wound healing.

Mechanism of crosslinking of proteins by glutaraldehyde III. Reaction with collagen in tissues

Bovine pericardium, a dense collagenous connective tissue, was crosslinked with glutaraldehyde using different modalities of fixation. The degree of crosslinking was evaluated as a function of the ability of CNBr and pronase to solubilize collagen. Our results suggest that glutaraldehyde fixes primarily the surface of the fibers and creates a polymeric network which hinders the further crosslinking of the interstitium of the fiber. When a low concentration of glutaraldehyde was used, a slow time-dependent crosslinking process was observed. This slow process is maintained over a long period of time, greatly beyond that required for the actual penetration of glutaraldehyde to occur.

The effect of aging on bone formation in rats: Biochemical and histological evidence for decreased bone formation capacity

SummaryEctopic bone formation by subcutaneously implanted demineralized bone matrix powder (DBM) was assessed biochemically and histologically in Fischer 344 rats of different ages. The total calcium accumulated in implants was greatly depressed in older rats, as was the rate of45Ca deposition. High alkaline phosphatase activity appeared later in the 10- and 16-month-old rats compared with 1-month-old rats, and the magnitude of the alkaline phosphatase activity was decreased in 16-month-old rats. The accumulation of the bone-specific vitamin K-dependent bone protein (bone gla protein, BGP) was decreased in the implants in older rats. Histological examination of the implants confirms the decreased ability of aged animals to produce bone in response to DBM. Measurements of total calcium, alkaline phosphatase, and BGP at the site of demineralized bone matrix implants clearly demonstrates that bone formation decreases dramatically with increasing age. Significant differences in total calcium can be detected even between 1-month-old and 3-month-old rats. Serum BGP shows a marked decrease (47%) between 1-month- and 3-month-old rats, a decrease not paralleled by a similar decrease in BGP present in calvarial or tibial bone.

Collagen shortening. An experimental approach with heat.

Decreasing joint laxity is a clinical goal of ligament reconstructions. This in vitro study examined the structural and histologic effects of heat shrinkage of human collagen. Two preliminary studies were performed to assess the effect of heat on fresh frozen human tendons obtained from a local tissue bank. As heat was applied to tissue in a saline solution, the percent shrinkage was plotted against temperature. A second study used a freebeam Nd:YAG laser to maximally shrink patellar tendons measuring percent shrinkage versus energy applied. Finally, the effects of 10% shrinkage of fresh frozen human patellar tendons were analyzed mechanically and histologically. Consistent tendon shrinkage curves were found with increasing temperatures in a saline solution. A sharp increase in shrinkage to approximately 70% of resting length was noted around 70° C. Tendon shrinkage by laser induced heat was precise and dose related. Tensile testing of the tendons shortened 10% of their resting length showed a decrease in load to failure to approximately ⅓ compared with that of historical control specimens. Histologic sections showed a well demarcated site of diffuse denaturation and degeneration of collagenous elements. Normal collagen was present adjacent to these thermal changes. These experiments showed that collagen tissue can be shortened precisely by the application of heat. Future studies need to examine the in vivo biologic response of shortened collagen tissue with time, especially recollagenization, restoration of length, and the long term biomechanical effects.

Mechanism of Crosslinking of Proteins by Glutaraldehyde II. Reaction with Monomeric and Polymeric Collagen

Collagen in three different states, i.e. native soluble molecules, denatured molecules and reconstituted fibers, was exposed to various concentrations of glutaraldehyde. The degree of intramolecular and intermolecular crosslink formation was evaluated by measuring collagen solubility, beta and gamma chain formation, resistance towards cleavage by CNBr or collagenase digestion. Modification of lysyl residues was measured by amino acid analysis. When dilute collagen solutions were reacted with low concentrations of glutaraldehyde, intramolecular crosslinks were observed as the predominant crosslinks. When the glutaraldehyde concentration was increased, the collagen became more insoluble, indicating the formation of intermolecular crosslinks. When reconstituted collagen fibers were reacted with low concentrations of glutaraldehyde, intermolecular crosslinks were formed, which prevented the material from being solubilized by CNBr. However, these materials could still be solubilized by collagenase. When the glutaraldehyde concentration was increased, the materials became resistant to collagenase, while there was only a small increase in the number of lysyl residues modified. This reflects an increase in the molecular length of the glutaraldehyde polymers extending from the initial glutaraldehyde and lysyl residue reaction sites rather than an increase in the actual number of crosslinking sites.

Differences in Collagen Metabolism between Normal and Osteoarthritic Human Articular Cartilage

Normal human articular cartilage synthesizes only one type of a chain, which exhibits the chromatographic behavior of the αl(II) chains described for chick and bovine cartilage. Osteoarthritic cartilage, on the other hand, synthesizes in addition a collagen containing α2 chains and β components. The different structural features of the two types of collagen may account for some of the functional defects of osteoarthritic cartilage.

Collagen types. Molecular structure and tissue distribution.

The collagens are products of a superfamily of closely related genes. Currently, there are 13 described collagens encompassing at least 25 separate genes. The collagen molecules can be categorized into four classes. Class I consists of molecules that form the banded collagen fibers that are readily seen by routine electron microscopy. The banded fibers are heterogenous with respect to collagen type, containing at least two and often three collagen types in each fibril. This multiplicity is believed to effect the rate of fibril growth and the final fibril diameter. Class II contains collagens that adhere to the surface of the banded fibrils. The function of these molecules is not yet known. The third Class consists of molecules that form independent fiber systems. These include the basement membrane, beaded filaments, anchoring fibrils, and the network surrounding hypertrophic chondrocytes. The last class contains several collagens with unknown fiber forms, and whose functions are unclear. Tissues contain multiple fiber forms and therefore many individual collagen types. Bone is no different, and there are presently four known collagens in the bone cortex. This article summarizes knowledge of the structures and functions of the collagen superfamily.

Quantitative and sensitive in vitro assay for osteoinductive activity of demineralized bone matrix

A sensitive, rapid, reliable and quantitative method to check the bone forming potential of demineralized bone matrix (DBM) has been developed. The osteoinductivity of the bone morphogenetic proteins (BMPs), present in DBM, can be measured in vitro using a pluripotent myoblast C2C12 cell line. Alkaline phosphatase activity induced by co‐incubation of DBM with C2C12 cells was dose‐responsive and corresponds to the amount of active BMPs in DBM. Bone forming potential was simultaneously tested in vivo by implanting DBM intra‐muscularly in nude rats. ALP activity induced in C2C12 cells, correlated with bone formation in vivo (r = 0.88), determined by alkaline phosphatase activity, mineralization density and histomorphology of the DBM explants. Results from DBM batches, originating from five established Bone Banks, showed good consistency between in vitro and in vivo assays. However, DBM activity varied widely from bank to bank as well as from batch to batch within the same bank.