Osteogenic differentiation of dura mater stem cells cultured in vitro on three-dimensional porous scaffolds of poly(epsilon-caprolactone) fabricated via co-extrusion and gas foaming.

A novel scaffold fabrication method utilizing both polymer blend extrusion and gas foaming techniques to control pore size distribution is presented. Seventy-five per cent of all pores produced using polymer blend extrusion alone were less than 50microm. Introducing a gas technique provided better control of pore size distribution, expanding the range from 0-50 to 0-350microm. Varying sintering time, annealing temperature and foaming pressure also helped to reduce the percentage of pore sizes below 50microm. Scaffolds chosen for in vitro cellular studies had a pore size distribution of 0-300microm, average pore size 66+/-17microm, 0.54+/-0.02% porosity and 98% interconnectivity, measured by micro-computed tomography (microCT) analysis. The ability of the scaffolds to support osteogenic differentiation for subsequent cranial defect repair was evaluated by static and dynamic (0.035+/-0.006ms(-1) terminal velocity) cultivation with dura mater stem cells (DSCs). In vitro studies showed minimal increases in proliferation over 28 days in culture in osteogenic media. Alkaline phosphatase expression remained constant throughout the study. Moderate increases in matrix deposition, as assessed by histochemical staining and microCT analysis, occurred at later time points, days 21 and 28. Although constructs cultured dynamically showed greater mineralization than static conditions, these trends were not significant. It remains unclear whether bioreactor culture of DSCs is advantageous for bone tissue engineering applications. However, these studies show that polycaprolactone (PCL) scaffolds alone, without the addition of other co-polymers or ceramics, support long-term attachment and mineralization of DSCs throughout the entire porous scaffold.

Mesenchymal stem cells from rat visceral fat exhibit multipotential differentiation in vitro.

Human subcutaneous fat-derived stem cells were recently shown to have the potential to differentiate in vitro into a variety of cell types, including adipocytes, osteoblasts, chondrocytes, and myoblasts (Zuk et al., Tissue Eng. 2001;7:211-228). Subcutaneous adipose tissue may therefore prove to be an easily acquired and abundant source of stem cells. Presently it is unclear whether mammals such as rats (which possess small or nonexistent subcutaneous fat pads) contain mesenchymal stem cells within the visceral fat of the abdominal cavity, or whether the visceral fat of any species contains stem cells. In this study we isolated and expanded a pool of mesenchymal cells from visceral fat of adult Sprague-Dawley rats and induced their differentiation in vitro into adipocytes, osteoblasts, neural cells, and chondrocytes. The differentiated phenotypes were verified by morphology as well as detection and expression of tissue-specific protein and mRNA. We conclude that despite well-documented differences in the metabolic and biochemical properties among anatomically distinct depots of fat, the visceral fat of rats contains adult mesenchymal stem cells with developmental potential similar to those isolated from subcutaneous fat in humans. Therefore, animals such as rats provide both a source of fat-derived stem cells and an immunocompetent, autologous host animal in which to investigate the capacity of the fat-derived cells to differentiate and form tissues in vivo.

Correction of coronal suture synostosis using suture and dura mater allografts in rabbits with familial craniosynostosis.

OBJECTIVE: Resynostosis following surgical correction of craniosynostosis is a common clinical correlate. Recent studies suggest that the dura mater is necessary to maintain suture patency. It has also been hypothesized that dura mater from synostotic individuals may provide aberrant biochemical signals to the osteogenic fronts of the calvaria, which result in premature suture fusion and subsequent resynostosis following surgery. This study was designed to test this hypothesis by surgically manipulating the coronal suture and dura mater in rabbits with familial craniosynostosis to prevent postsurgical resynostosis. DESIGN: Craniofacial growth and histomorphometric data were collected from 129 rabbits: 72 normal controls and 57 rabbits with bilateral coronal suture synostosis (15 unoperated on controls; 13 surgical controls; 9 dura mater transplant only; 10 suture transplant only; and 10 suture and dura mater transplant). At 10 days of age, all rabbits had radiopaque amalgam markers placed on either side of the coronal, frontonasal, and anterior lambdoidal sutures. At 25 days of age, 42 synostosed rabbits had a 3 to 5-mm wide coronal suturectomy. Coronal sutures and/or underlying dura mater allografts were harvested from same-aged, wild-type, isohistogenic control rabbits and transplanted onto the dura mater of synostosed host rabbits. Serial radiographs were taken at 10, 25, 42, and 84 days of age, and the suturectomy sites were harvested at 84 days of age in 44 rabbits and serially sectioned for histomorphometric examination. RESULTS: Results revealed that cranial vault growth was significantly (p < .05) improved following surgical release of the fused coronal suture compared with synostosed rabbits who were not operated on but was still significantly different (p < .05) from that of normal control rabbits. By 84 days of age, significant (p < .05) differences were noted in calvarial suture marker separation, cranial vault shape indices, and cranial base angles between rabbits with and without dura mater allografts, probably as a result of resynostosis of the suturectomy site or suture-only allografts. Qualitative histological examination revealed that at 84 days of age rabbits with suture and dura allografts had patent coronal sutures, suture-only allografts had fused coronal sutures with extensive endosteal hyperostosis, dura mater-only allografts had some new bone in the suturectomy site that resembled rudimentary osteogenic fronts, and suturectomy controls had extensive endosteal bone formation and resynostosis of the suturectomy site. Significantly (p < .05) more bone was found in the suturectomy sites of rabbits without dura mater allografts compared with rabbits with dura mater allografts. CONCLUSIONS: Results support the initial hypothesis that normal dura mater allografts will maintain suture or suturectomy site patency and allow unrestricted craniofacial growth. However, it is still unclear whether the dura mater from normal rabbits was providing biochemical signals to the transplanted sutures or suturectomy sites or simply acting as a barrier to prevent abnormal biochemical signals from the dura mater of synostosed rabbits from reaching the calvaria. The clinical and therapeutic implications of these procedures are discussed.

Cranial suture obliteration is induced by removal of transforming growth factor (TGF)-beta 3 activity and prevented by removal of TGF-beta 2 activity from fetal rat calvaria in vitro.

Cranial suture morphogenesis requires soluble, heparin-binding factors secreted by the dura mater to resist premature osseous obliteration. Elevated levels of transforming growth factor (TGF)-beta 1, TGF-beta 2, and TGF-beta 3 have previously been noted in cranial sutures undergoing normal and premature sutural obliteration. To examine the role of TGF-beta s in regulating cranial suture morphogenesis, an established in vitro, serum-free, calvarial culture system was used. In this system, fetal rat coronal sutures undergo apparently normal suture morphogenesis in the presence of dura mater, but undergo osseous obliteration in the absence of dura mater. Neutralizing polyclonal antibodies to TGF-beta 1, TGF-beta 2, or TGF-beta 3 were added to cultures of fetal day 19 rat calvaria, which were harvested at 3, 4, or 5 days, processed for histology, sectioned, and examined. Coronal sutures from calvaria cultured in the presence of dura mater resisted obliteration, either alone or in the presence of TGF-beta 1 or TGF-beta 2 neutralizing antibodies. However, sutures from calvaria cultured in the presence of TGF-beta 3 neutralizing antibodies became obliterated. Conversely, sutures from calvaria cultured in the absence of dura mater became obliterated by bone, either alone or in the presence of neutralizing antibodies to TGF-beta 1 or TGF-beta 3. However, those sutures cultured in the presence of neutralizing antibodies to TGF-beta 2 were rescued from osseous obliteration.

Repair of a rodent nasal critical-size osseous defect with osteoblast augmented collagen gel.

OBJECTIVES/HYPOTHESIS: Facial skeletal defects are a common challenge for the otolaryngologist. Type I collagen gels have shown promise in the repair of nonhealing critical size defects (CSDs) of facial bone by providing scaffolding for new bone growth by osteoblasts at the defect perimeter. The objective of the present study was to evaluate the effect that suspending osteoblasts within a type I collagen gel has on the repair of a rodent facial CSD. STUDY DESIGN: Randomized controlled trial using a rodent model. METHODS: A previously described facial CSD was created by removing the nasalis bones with a cutting burr to the level of the nasal mucosal membranes on 18 Sprague-Dawley rats. Groups of six animals were treated with an implant containing either 300 microg of type I collagen gel, 12 x 10(5) osteoblasts suspended within type I collagen gel, or 12 x 10(5) fibroblasts suspended within type I collagen gel for comparison. After 30 days the animals-were examined at necropsy with planimetry, histological analysis of new bone growth, and radiodensitometric analysis of bone thickness. RESULTS: All animals had complete coverage with a thin layer of bone. Histological sectioning revealed an increased thickness in the osteoblast augmented group. Radiodensitometric measurements revealed a statistically significant increase in bone repair in the osteoblast group compared with the collagen-only group (P < or = .0005) and the fibroblast group (P < or = .04). CONCLUSION: Type I collagen gels augmented with an osteoblastic suspension significantly enhance the repair of nasal CSDs in a rodent model. The use of cultured bone precursor cells represents a leap forward in osteoengineering.

Repair of an osseous facial critical-size defect using augmented fibrin sealant.

OBJECTIVE: Osseous defects of the head and neck are a common challenge for the otolaryngologist. To develop improved reconstructive options, osteoconductive engineering experiments are being conducted. A nasal critical-size defect (CSD) model has previously been described in which less than 7% bone healing is observed over 6 months. An implant containing fibrin sealant with and without osteoprogenitor cells is evaluated in this model. STUDY DESIGN: Randomized controlled trial using a rodent model. METHODS: A nasal CSD was surgically created in 18 male retired breeder Sprague-Dawley rats. Six animals were not implanted with any material, six received fibrin sealant consisting of fibrin (25 mg/mL) and thrombin (1000 U/mL), and six were implanted with fibrin sealant and rat calvarial osteoprogenitor cells (1.8 x 10(6) cells/mL). Thirty days later, the animals were examined at necropsy by planimetry, histological analysis of new bone growth, and radiodensitometric analysis of bone thickness. RESULTS: A thin layer of bone covered the defect in all of the treated animals. A statistically significant increase in bone density (P < .05) between fibrin sealant plus osteoprogenitor cells and each of the other groups was shown using radiodensitometric analysis. Histological analysis also confirmed this difference. CONCLUSION: Osteoprogenitor cells contained within fibrin sealant result in a greater augmentation of bone regeneration than controls or fibrin sealant alone.

Insulinlike growth factor 1- and 2-augmented collagen gel repair of facial osseous defects.

BACKGROUND: Defects of the facial bone structure are common problems for the facial plastic surgeon. Native type 1 collagen gels (T1CGs) have been shown to mediate repair of facial critical-size defects in rat models. OBJECTIVE: To evaluate the efficacy of T1CG augmented with insulinlike growth factor (IGF) 1, IGF-2, and a combination of IGF-1 and IGF-2 on the repair of facial critical-size defects in a rodent model. METHODS: Twenty-four retired male breeder Sprague-Dawley rats were divided into 4 groups of 6 animals. Facial critical-size defects were created by removing the nasalis bones with a bone-cutting drill. Defects were treated with 300 pg of type 1 collagen gel (T1CG), T1CG augmented with 3 microg of IGF-1, T1CG augmented with 3 microg of IGF-2, or T1CG augmented with a combination of 3 microg of IGF-1 and 3 microg of IGF-2. After 30 days the animals were examined at necropsy with precise planimetry, histological analysis of new bone growth, and radiodensitometric analysis of bone thickness. RESULTS: Radiodensitometric measurements showed that IGF-2 augmentation resulted in greatest osseous healing, with measurements being statistically significant over those of all other groups (P< or = .03). Combination IGF-1 and IGF-2 had osseous healing that was intermediate between IGF-1 augmentation and IGF-2 augmentation alone, with measurements being statistically significant over those of unaugmented gels (P<.001) and IGF-1 augmentation (P< or = .03). Augmentation with IGF-1 resulted in healing that was significant over that of unaugmented gels (P< or = .04). CONCLUSION: Collagen gels augmented with IGF significantly enhance the osteoconductive repair of nasal critical-size defects in a rodent model, with IGF-2 showing highest efficacy.

Repair of nasal defects using collagen gels containing insulin-like growth factor 1.

OBJECTIVE: Facial osseous defects are a common and challenging problem for the otolaryngologist-head and neck surgeon. Current methods of repair including synthetic grafts, cadaveric material, and autologous tissue have drawbacks of foreign body reactions, infectious agent transmission, and the morbidity of a second surgical site. In the effort to develop an ideal technique for osseous reconstruction, a critical-size facial defect has previously been developed in the Sprague-Dawley rat. This model exhibits less than 10% healing by surface area over 6 months. A novel approach to osseous reconstruction is attempted using this model with type I collagen gel augmented with insulin-like growth factor 1 (IGF-1). STUDY DESIGN: Randomized controlled trial using a rodent model. METHODS: Twelve adult male Sprague-Dawley rats underwent a surgical procedure to produce a critical-size nasal defect by removing the nasal bones with a cutting burr. Six animals were repaired with 300 microg of type I collagen gel. Six animals were repaired with 300 microg of type I collagen gel augmented with 3.0 microg of IGF-1. Thirty days later, the animals were examined after necropsy. Precise planimetry, radiodensitometric analysis, and histologic sectioning were performed. RESULTS: All animals had complete coverage of this defect with a thin layer of bone. Radiodensitometric analysis indicated that there was a statistically significant (P < .037) increase in bone density in the collagen plus IGF-1 group compared with that of collagen only. In addition, histologic evaluation revealed increased bone density and thickness in the IGF-1 group. CONCLUSION: Type I collagen gel augmented with IGF-1 results in a significant increase in healing of a nasal critical-size defect in a rodent model.

Dura mater maintains rat cranial sutures in vitro by regulating suture cell proliferation and collagen production.

Craniosynostosis, the premature osseous obliteration of cranial vault sutures, can result from mutations in genes encoding components of growth factor signaling systems or the extracellular matrix (ECM). Little is known of the capacity of osteoprogenitor cells of the cranial sutures to divide or to synthesize ECM in situ. Osteoblasts derived from patients with prematurely fused sutures were reported to express alkaline phosphatase and osteocalcin at elevated levels, while proliferating at a rate comparable to control cells [DePollack et al., JBMR, 1996]; however, the suture osteoprogenitors, the population most likely to show proliferative abnormalities, were not present in the fused sutures used for this study. A model in which rat coronal sutures and associated bones develop normally in vitro, but in which sutures can be induced to fuse in the absence of dura mater, was used to examine cell proliferation and total protein synthesis in unfused sutures cultured in the presence of dura mater or in sutures induced to fuse in the absence of dura mater. Significantly increased cell proliferation was seen in suture cells prior to sutural obliteration, which returned to control levels as sutural fusion proceeded. Collagen synthesis in fusing sutures was elevated compared to non-fusing sutures and comparable to that seen in bone. Results indicated that in the absence of intercellular signals provided by the dura mater, suture cell proliferation increased initially, followed by increased synthesis of collagenous ECM within the suture and subsequent osseous obliteration of the suture. Thus factors originating in the dura mater affected suture cell proliferation and ECM production and were required for the maintenance of suture patency.

Elevated levels of transforming growth factors beta 2 and beta 3 in lambdoid sutures from children with persistent plagiocephaly.

OBJECTIVE: To analyze the pertinent history and physical findings specific to the subset of patients with a progressive posterior skull deformity, requiring surgery to correct their deformity. PATIENTS: Since the Academy of Pediatrics issued its recommendation on supine positioning of infants to prevent sudden infant death syndrome (SIDS) in 1992, 73 children have presented to the University of Virginia Craniofacial Anomalies Clinic with posterior-skull deformities. The majority were successfully managed with conservative therapy, but in six patients, the deformity was severe and persistent, requiring surgical correction. All six children were older (7.5-12 mo), presenting with more severe morphologic appearances and a higher incidence of associated neurodevelopmental delay. Three had family backgrounds of isolated craniosynostosis. METHODS: Characteristics of these patients were examined to determine why they may have differed from those that responded to conservative management. Immunohistochemical staining of their lambdoid sutures was performed. RESULTS: Significantly increased staining for TGF-beta 2 and TGF-beta 3, potent stimulators of bone cell growth and differentiation, was seen in all 'affected' sutures from the flattened side of the skull, compared to unaffected sutures from the protruding side of the skull-a pattern similar to that seen during normal bony obliteration of calvarial sutures. CONCLUSION: The majority of patients with posterior plagiocephaly associated with positioning responded to conservative management, while a small subset of patients with persistent posterior skull deformation required surgical intervention. A genetic basis for the latter patients' persistent plagiocephaly, rather than positioning, cannot be ruled out. Genetics, prolonged external pressure against the sutures, or a combination of these factors may lead to permanently raised levels of growth factors in 'affected' sutures.

Laminin E8 alveolarization site: heparin sensitivity, cell surface receptors, and role in cell spreading.

Cell adhesion to amino acids 2179-2198 (SN-peptide) of the laminin-1 alpha1-chain is required for lung alveolar formation in vitro (M. L. Matter and G. W. Laurie. J. Cell Biol. 124: 1083-1090, 1994). The nature of the SN-peptide receptor(s) was probed with neutralizing anti-integrin monoclonal antibodies (MAb), cells lacking integrin subunits, soluble heparin, and SN-peptide columns. Cell adhesion and spreading studies confirmed the specificity of SN-peptide and revealed adhesion to be unaffected by inclusion of anti-beta1-, anti-alpha(2-6)- or anti-alpha(V)beta5-integrin MAb. Cells lacking beta1- or alpha6-integrin subunits were fully adherent. Adhesion was heparin, but not chondroitin sulfate or heparinase, sensitive, much as is alpha-dystroglycan-laminin-1 binding. Heparin eluted approximately 155- and 180-kDa cell-surface proteins from SN-peptide columns. An additional approximately 91-kDa protein was eluted by EDTA. All were unrecognized by anti-beta1-integrin MAb. SN-peptide therefore interacts with three cell-surface proteins for which the identity remains to be determined.

TGF-beta 1, TGF-beta 2, and TGF-beta 3 exhibit distinct patterns of expression during cranial suture formation and obliteration in vivo and in vitro.

Cranial sutures function as bone growth centers while themselves remaining unossified. Rat frontonasal sutures become obliterated by neonatal day 21 (N21), while coronal sutures do not fuse over the life of the animal. Coronal sutures induced to undergo osseous obliteration in vitro after removal of the dura mater were found to require soluble, heparin-binding factors present in dura mater to resist osseous obliteration. Transforming growth factor beta 1 (TGF-beta 1), beta 2, and beta 3, heparin-binding factors known to regulate bone cell proliferation and differentiation, were considered likely candidates. The presence and distribution of these factors in calvarial tissues both in vivo and in vitro were established by immunohistochemical analysis, while reverse transcription followed by polymerase chain reaction (RT/PCR) was employed to determine the presence of transcripts for these factors in mRNA isolated from microdissected dura mater. Results indicated that the presence of TGF-beta 1 and TGF-beta 2 were associated with developing coronal and frontonasal sutures, and that the continued presence of these factors was associated with osseous obliteration of the frontonasal suture. However, increased TGF-beta 3 immunoreactivity was associated with the coronal suture remaining unossified. RT/PCR demonstrated the presence of transcripts for TGF-beta 1, beta 2, and beta 3 in dural tissues isolated from rat calvaria. These data support the notion of a role for TGF-beta s in regulating cranial suture morphogenesis and establish the in vitro model as a valid system for examining mechanisms by which growth factors regulate both suture morphogenesis and bone growth at the suture site.

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.

Altered production of laminin and nidogen by high and low metastatic variants of murine melanoma cells.

An altered ability to interact with and degrade extracellular matrix molecules is a common feature of the malignant phenotype. Although changes in the expression of matrix proteins in metastases in vivo are relatively well documented, little is known about the changes in matrix production by malignant cells in culture. Here we have examined the synthesis of the basement membrane components laminin and nidogen (entactin) by low and high metastatic variants of the K-1735 murine melanoma cells. Protein deposition was examined by western blotting as well as immunofluorescence; protein synthesis was examined by immunoprecipitation with specific antibodies. Gene expression was also evaluated by measuring steady-state mRNA levels using cDNA probes on northern and dot-blots. Laminin gamma 1 levels appeared to be similar in both high and low metastatic lines; however, the high metastatic lines had reduced levels of the laminin beta 1 chain. On the contrary, nidogen expression was observed only in the high metastatic lines. Traces of a laminin alpha chain were present only in immunoprecipitates of the low metastatic cells and could not be detected in the high metastatic cells. Both high and low metastatic cells deposited an extracellular matrix of basement membrane components, with laminin deposition decreased in high metastatic cells. Modified expression, production, and deposition of basement membrane components in high metastatic melanoma cells could be involved in their altered interactions with the extracellular matrix.

A rapid in vitro assay of cellular chemomigration in an epithelial carcinoma cell line.

We have studied the chemomigration activity of an epithelial carcinoma cell line using a modified 96-well Boyden chamber apparatus consisting of upper and lower wells separated by an 8-microns pore polycarbonate filter. Cells from the malignant squamous carcinoma cell line A-431 were plated in the upper wells over a collagen IV-coated filter. In chemokinesis assays, the cells were allowed to migrate toward NIH 3T3 fibroblast-conditioned medium or control media in the lower wells for 6 hours at 37 degrees C with 10% CO2. A-431 cells preferentially migrate across the barrier toward conditioned media but not control media. Control normal keratinocytes showed no migration. A highly metastatic melanoma cell line and poorly metastatic melanoma cell line, in which chemomigration has been shown previously to correlate with metastatic potential, were used as positive and negative cellular controls. This system provides a rapidly quantifiable method by which the invasion characteristics of multiple cell lines can be studied simultaneously in a single assay using the 96-well format.

Cranial sutures require tissue interactions with dura mater to resist osseous obliteration in vitro.

A chemically defined serum-free medium, which supports the development of bones and fibrous tissues of rat calvaria from nonmineralized mesenchymal precursor tissues, was employed to investigate tissue interactions between the dura matter and overlying tissues. Fetal calvarial rudiments from stages prior to bone and suture morphogenesis (fetal days 19 and 20) and neonatal calvarial rudiments with formed sutures (day 1) were cultured with and without associated dura mater. Removal of calvaria for in vitro culture allowed the examination of suture morphogenesis in the absence of tensional forces exerted on the sutures via fiber tracts in the dura mater originating in the cranial base. Ossification of frontal and parietal bones proceeded in a fashion comparable to development in vivo, but the cranial (coronal) sutures--primary sites for subsequent skull growth--were obliterated by osseous tissue union in the absence of dura mater. Bony fusion did not occur when rudiments were cocultured with dura mater on the opposite sides of 0.45 microns polycarbonate transwell filters, suggesting that the influence of dura mater on sutural obliteration was mediated by soluble factors rather than cell-cell or cell-matrix interactions. These results indicate that cell signaling mechanisms rather than biomechanical tensional forces are required for morphogenesis of the calvaria.

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.

Laminin and fibronectin content of mouse glomerular and tubular basement membrane.

Because the glomerular basement membrane (GBM) is subject to damage in a multitude of renal diseases, a model of basement membrane permeability properties would be useful for learning more about this important barrier. Isolated, perfused tubular basement membrane (TBM) allows measurement of permeability, but it is not known whether TBM is similar enough to GBM for data to be extrapolated from this model to the glomerulus. As a first approach to assessing differences between GBM and TBM, we looked at composition. Renal glomeruli and tubules were isolated from Swiss-Webster mice by sucrose-gradient centrifugation. GBM and TBM were isolated by sonication in 1% deoxycholate and then subjected to a sequential extraction procedure. Analysis of the solubilized basement membranes by electrophoresis revealed a complex mixture of proteins. Immunoblot analysis demonstrated that, among the proteins, laminin and fibronectin were found exclusively in the guanidine and guanidine/dithiotreitol extracts. The total amount of laminin extracted in GBM, 1.8 +/- 0.001 micrograms/mg dry weight (n = 2 groups animals, by inhibitory ELISA), was significantly less than in TBM, 3.4 +/- 0.1 micrograms/mg dry weight (n = 2); however, the total amount of fibronectin extracted did not differ between GBM and TBM, 8.2 +/- 0.8 and 7.7 +/- 1.0 micrograms/mg dry weight (n = 2) respectively. Examination of deoxycholate supernatants was carried out to see if components of GBM or TBM were solubilized during isolation of basement membranes. Immunoblot analysis revealed loss of some laminin and fibronectin occurred during the detergent isolation of GBM and TBM. We conclude that GBM and TBM are qualitatively similar in that they have the same protein components, but differ significantly in content of laminin and probably other macromolecular components.

In the absence of periosteum, transplanted fetal and neonatal rat coronal sutures resist osseous obliteration.

Normal craniofacial development depends on expansion of the cranial vault by growth at the sutures. Inappropriate development of the sutures leads to global disruption of patterns of craniofacial growth. Tissue interactions between dura mater and suture matrix play a critical role in the phenotypic maintenance of cranial sutures. However, the function of the periosteum in this process remains under-reported and controversial. To examine the contribution of periosteum in maintaining the patency of coronal sutures, fetal and neonatal rat coronal sutures were transplanted to surgically created defects in adult rat host parietal bones. These sutures were examined for their ability to persist in the host milieu in the presence and absence of both donor and host periosteum. This study established that removal of both host and transplant periosteum, unlike removal of dura mater, did not lead to obliteration of either fetal or neonatal sutures. Thus, periosteum and dura mater are nonequivalent tissues with respect to influence on suture patency.