Utilization of type I collagen gel, demineralized bone matrix, and bone morphogenetic protein-2 to enhance autologous bone lumbar spinal fusion.

Autologous bone grafts are currently considered "gold standard" material for achieving long-term spinal arthrodesis. The present study was performed to determine whether demineralized bone matrix (DBM), type I collagen gels, or bone morphogenetic protein-2 (BMP-2) can improve autologous bone spinal fusions. Using a unilateral decompression-contralateral fusion technique in dogs, each of these materials was added to an autologous bone graft. Volumetric analysis, histological analysis, and biomechanical testing were performed to assess the effectiveness of each material. The DBM had an inhibitory effect on solid bone fusion of the spine, whereas the type I collagen gels improved the bony interface between the graft and the host spine. The BMP-2 strongly enhanced the amount of bone deposition at the fusion site and increased the number of intervertebral levels that were solidly fused. This study strongly supports the use of BMP-2 as an additive to autologous bone grafts in spine stabilization.

Nasal reconstruction using an osteoconductive collagen gel matrix.

BACKGROUND: Congenital malformations, granulomatous diseases, and trauma can all cause destruction of the nasal structural framework, resulting in functional nasal obstruction and altered facial cosmesis. Current methods of nasal reconstruction include cartilaginous and bony grafts, Silastic implants, and homograft only materials. However, these techniques have significant functional and cosmetic drawbacks and are not risk free. Native, isotonic, neutral-pH, space-filling type I collagen gels have been shown to mediate total repair of critical-size collagen grafts provide a framework for rapid intramembranous ossification and osteoconduction of bone from the perimeter of a defect, resulting in total bony coverage. OBJECTIVE: To evaluate a novel approach to nasal reconstruction using a major defect of the bony nasal dorsum with a type I collagen gel matrix. DESIGN: Sixteen retired male breeder Sprague-Dawley rats were divided into control and experimental groups. The nasal bones were exposed through a dorsal incision and completely removed with a bone-cutting drill to the level of the mucosal membranes of the nasal vestibule. Defects in the experimental animals were then implanted with 200 micrograms of type I collagen gel, with control animals receiving no inlay. After 6 weeks, the animals were examined with three-dimensional computed tomography before necropsy, at which time the defects were photographed, measured by planimetry, and sectioned for histologic analysis. RESULTS: Experimental defects were observed to manifest 100% surface area healing with a thin layer of bone using a type I collagen gel osteoconductive implant for nasal reconstruction. Conversely, control animals showed only a 5.7% (+/- 3.7% SD) healing by area. Histologic sections of the collagen gel implant revealed restoration of the anatomy with a thin plate of immature bone spanning the defect in continuity with the cartilage of the nasal septum and with apparent preservation of maxillonasalis suture lines. CONCLUSIONS: Native, isotonic, neutral-pH, space-filling collagen gels positively influenced the repair of large nasal defects, which showed minimal bone closure in untreated animals. Their use in this role merits further investigation.

Repair of critical size rat calvarial defects using extracellular matrix protein gels.

In this study the authors examined the capacity of gels of reconstituted basement membrane, laminin, and type I collagen to mediate repair of critical size defects in rat calvaria. Although autografts are widely used to repair bone defects caused by trauma or surgical treatment of congenital malformations, neoplasms, and infections, an adequate quantity of graft is not always available. Allogenic bone is readily available, but its use is associated with an increased incidence of nonunion, fatigue fracture, and rejection. Biologically active, purified components of basement membranes, which have been shown to promote osteogenic differentiation and angiogenesis in vitro and type I collagen (the major constituent of bone extracellular matrix) can be formed into native isotonic space-filling gels. In this study critical size calvarial defects were created in retired male Sprague-Dawley rats. Thirty-six animals were divided into seven groups. Group 1 (control) received no treatment for the defects. Group 2 animals were implanted with methylcellulose. Groups 3, 4, 5, and 6 were implanted with gels of type I collagen, reconstituted basement membrane, or laminin, respectively. The last group of three animals (Group 7) was implanted with 100 micrograms of type I collagen gels (identical to Group 3) and sacrificed at 20 weeks following a single CT scan to determine if complete healing could be obtained with this method given sufficient time. Except for rats in the type I collagen group that was evaluated by multiple computerized tomography (CT) scans biweekly from 2 to 12 weeks, bone repair was evaluated using CT at 12 weeks. Healing was quantified using three-dimensional reconstruction of CT. Following the final CT scan in each experimental group, animals were sacrificed, and a sample of tissues was evaluated by conventional histology. Animals treated with type I collagen gels showed 87.5% repair of the area of the defects at 12 weeks and 92.5% repair by 20 weeks. Increasing the gel volume 1.5 x accelerated complete repair to 3 months. Murine-reconstituted basement membrane and laminin gels induced 55.5% and 46.3% repair, respectively, at 3 months. In untreated control animals 7% repair of the area of the defects showed at 3 months. Histological analysis confirmed new bone formation in partial and completely healed defects. Bioengineered native collagen gels may have wide applicability for bone repair as an alternative bone graft material alone, in combination with autograft or marrow aspirate, or as a delivery system for osteogenic growth factors.

Tissue interactions with underlying dura mater inhibit osseous obliteration of developing cranial sutures.

Cranial sutures play a critical role in calvarial morphogenesis, serving as growth centers during skull development. Both biomechanical tensile forces originating in the cranial base and biochemical factors present in dura mater have been postulated as determinants of suture morphogenesis and patency. A rat transplant model free of the putative biomechanical influence of the dura and cranial base was used to investigate the role of the dura mater in both the initial morphogenesis and maintenance of sutures during skull growth. Day 19 fetal presumptive (F19) and day 1 neonatal differentiated (N1) coronal sutures, including associated frontal and parietal bones, were transplanted with or without underlying dura mater to the center of adult parietal bones. After 1, 2, and 3 weeks, transplanted tissues were examined histologically and histomorphometrically to determine whether sutures formed and whether they were obliterated by ossification in the absence of dura mater. Both F19 and N1 sutures remained patent for 2 weeks either in the presence or the absence of transplant dura mater. However, at 3 weeks, in the absence of transplant dura mater, sutures were obliterated by bone, while in the presence of dura mater sutures resisted ossification, demonstrating an essential requirement for interactions with the transplant dura mater in maintaining functional sutures. Both F19 and N1 transplants showed comparable bone growth (cross-sectional surface area), regardless of the presence of transplant dura mater. These experiments suggest that tissue interactions of a biochemical nature, rather than biomechanical forces generated through the cranial base, are required to maintain the suture as a non-ossified growth center. Furthermore, while the presence of dura mater was essential for maintenance of suture patency, fetal dura mater was not required for initial suture formation.

Malignant transformation of NIH-3T3 cells after subcutaneous co-injection with a reconstituted basement membrane (matrigel).

NIH-3T3 cells are non-tumorigenic when injected into athymic mice. If these cells are mixed with an extract of basement-membrane proteins (matrigel) and injected s.c., they form locally invasive and highly vascularized tumors. Cells cultured from the NIH-3T3-matrigel-induced tumors showed a transformed phenotype and lacked contact inhibition. When cultured in a gel of matrigel, they proliferated and formed branched and invasive colonies. In contrast, the parental NIH-3T3 cells cultured on matrigel remained as cell aggregates and were not invasive. I.V. injections of the tumor-derived NIH-3T3 cells produced many colonies on the surface of the lungs, whereas the parental NIH-3T3 cells were not metastatic. Zymographic analysis of the conditioned media obtained from both the tumor-derived and parental NIH-3T3 cells demonstrated higher amounts of the 72-kDa gelatinase (type-IV collagenase) enzyme in the tumor-derived cells. Also, tumor-derived NIH-3T3 cells, but not parental NIH-3T3 cells, secreted the 92-kDa type-IV collagenase. These studies suggest that the interaction of pre-malignant NIH-3T3 cells with extracellular matrix components may contribute to the process of tumor progression.

The view and responsibility of the advertising agency toward advertising claims.

Health care advertising agencies must create communication that is simultaneously creative, scientifically accurate, relevant, within fair balance, simple, motivating, within approved labeling, and approvable by the client, the dental profession, and regulatory bodies. The advertising must also stimulate sales of the product being advertised. Subjective decisions concerning these requirements place the agency and its client at risk for financial losses and embarrassment.

Identification of a 110-kDa nonintegrin cell surface laminin-binding protein which recognizes an A chain neurite-promoting peptide.

Laminin is a potent promoter of neurite outgrowth, and a synthetic peptide of 19 amino acids, PA22-2, from the A chain has been found to promote process formation. Using peptide affinity chromatography, we have identified a 110-kDa, cell surface ligand from both neural cells and brain which binds this sequence. This binding protein does not share immunological identity with the B1 chain of integrin, and reduction does not alter its mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Antibody to the 110-kDa protein stained cellular processes in vivo. Sequence analysis of the first 18 amino acids from the amino terminus yielded almost exact sequence identity with nucleolin, a major 110-kDa nucleolar phosphoprotein. Antibody to nucleolin, however, does not interact with the neural-derived, laminin-peptide-binding 110-kDa protein. The 110-kDa protein appears to be a ligand for a specific site on laminin.

Basement membrane and the SIKVAV laminin-derived peptide promote tumor growth and metastases.

Laminin, the major glycoprotein component of basement membrane, promotes the malignant phenotype. Cells which are adherent to laminin are more malignant than the non-adherent cells and in certain tumor cells, the number of laminin receptors is positively correlated with malignancy. Laminin also increases collagenase IV activity, an enzyme demonstrated to be critical for tumor spread. A site on laminin, containing the amino acid sequence SIKVAV, has been identified which when injected intravenously with B16F10 melanoma cells, causes an increase in the number of colonies on the surface of the lungs. This peptide does not affect tumor cell arrest in the vasculature or the immune system. It does promote angiogenesis in various in vitro and in vivo models, thereby facilitating tumor cell survival. When a complex mixture of laminin-enriched basement membrane components (Matrigel) is coinjected with tumor cells subcutaneously, tumor incidence and growth increases. Various tumor cell lines and primary isolates, which previously could not form tumors in mice, can be induced to grow rapidly in the presence of Matrigel. Slowly growing tumors or arrested tumors can also be induced to grow more quickly with additional injections of Matrigel. When an SIKVAV-containing synthetic peptide is coinjected with B16F10 tumor cells and Matrigel subcutaneously in mice, larger tumors are formed than that observed with either Matrigel or cells alone. Such studies define the role of laminin in tumor growth and spread and generate new models for studying therapeutic agents. Of particular interest is the ability to grow primary isolates which generally do not grow in mice.

Laminin potentiates differentiation of PCC4uva embryonal carcinoma into neurons.

The embryonal carcinoma PCC4uva differentiates into neurons in response to treatment with retinoic acid and dbcAMP. We used this in vitro model system to study the effects of laminin on early neural differentiation. Laminin substrata markedly potentiate neural differentiation of retinoic acid and dbcAMP-treated cultures. Only laminin induced more rapid neural cell body clustering, neurite growth and neurite fasciculation as compared to type IV collagen, type I collagen, and fibronectin substrata. Exogenous laminin substrata promoted greater cell attachment, cellular spreading and growth to confluence than type IV collagen, type I collagen, fibronectin and glass substrata. Laminin-induced effects were inhibited by addition of laminin antibodies or the synthetic laminin-derived peptide Ile-Gly-Ser-Arg-NH2 (YIGSR-NH2). Treatment with YIGSR-NH2 also inhibited neural differentiation in the absence of exogenous laminin substrata, whereas synthetic peptides containing the RGD sequence and a control peptide YIGSK-NH2 showed no inhibitory effects. These results are consistent with the hypothesis that specific interactions between an early differentiating cell population(s) and extracellular laminin are required during neural differentiation.

In vitro differentiation of neuron-like cells.

The pluripotent embryonal carcinoma, PCC4uva, differentiates into multiple cell types under standard culture conditions. Neuron-like cells first appear at 8 days of culture, and by Day 15 they represent approximately 3% of the cell population. We promoted neural differentiation by treatment with retinoic acid and dibutyryl cyclic adenosine monophosphate. Under these optimized culture conditions, neuron-like cells appear on the third day of culture, and by the sixth day, neuron-like cells represent 40% of the total cell population. After 10 days, the neuron-like cells represent approximately 60-80% of total cells. At this time the cells form large clusters of cell bodies that are interconnected by neurite fascicles. The cells express the neuron-specific proteins; Map2, Tau, neurofilaments, and the type III beta-tubulin isotype. We have recently used this developing system to investigate the effects of laminin substrates on neural differentiation.

Laminin receptors for neurite formation.

Laminin, a basement membrane glycoprotein promotes both cell attachment and neurite outgrowth. Separate domains on laminin elicit these responses, suggesting that distinct receptors occur on the surface of cells. NG108-15 neuroblastoma-glioma cells rapidly extend long processes in the presence of laminin. We report here that 125I-labeled laminin specifically binds to these cells and to three membrane proteins of 67, 110, and 180 kDa. These proteins were isolated by affinity chromatography on laminin-Sepharose. The 67-kDa protein reacted with antibody to the previously characterized receptor for cell attachment to laminin. Antibodies to the 110-kDa and 180-kDa bands demonstrated that the 110-kDa protein was found in a variety of epithelial cell lines and in brain, whereas the 180-kDa protein was neural specific. Antibodies prepared against the 110-kDa and 180-kDa proteins inhibited neurite outgrowth induced by the neurite-promoting domain of laminin, whereas antibodies to the 67-kDa laminin receptor had no effect on neurite outgrowth. We conclude that neuronal cells have multiple cell-surface laminin receptors and that the 110-kDa and 180-kDa proteins are involved in neurite formation.

The Personality Research Form as a therapy outcome measure of social behavior.

Reported two studies that examined the efficacy of the Personality Research Form (PRF) as an outcome measure for interventions designed to increase social competence. In the first study (N = 83), the degree to which PRF scales can predict criteria frequently used in social skills training outcome research was examined, and the PRF was shown to be sensitive to such criteria. The second study (N = 24) assessed the relative degree to which the PRF scales and more frequently used outcome measures, including the above criteria, are affected by factors non-specific to social skills training interventions (suggestion for improvement). While three PRF scales were affected significantly, demand effects were much more pervasive on the other measures, which suggests that those measures can be used to obtain valid estimates of treatment effects only when experimental control of non-specific effects is possible. Two PRF scales, Affiliation and Exhibition, were shown to be both sensitive to criteria and resistant to demand effects, and as such may be useful as outcome measures in non-controlled clinical settings.