Zone-Dependent Architecture and Biochemical Composition of Decellularized Porcine Nasal Cartilage Modulate the Activity of Adipose Tissue-Derived Stem Cells in Cartilage Regeneration.

Previous anatomical studies have shown different functional zones in human nasal septal cartilage (NC). These zones differ in respect to histological architecture and biochemical composition. The aim of this study was to investigate the influence of these zones on the fate of stem cells from a regenerative perspective. Therefore, decellularized porcine septal cartilage was prepared and subjected to histological assessment to demonstrate its equivalence to human cartilage. Decellularized porcine NC (DPNC) exposed distinct surfaces depending on two different histological zones: the outer surface (OS), which is equivalent to the superficial zone, and the inner surface (IS), which is equivalent to the central zone. Human adipose tissue-derived stem cells (ASCs) were isolated from the abdominal fat tissue of five female patients and were seeded on the IS and OS of DPNC, respectively. Cell seeding efficiency (CSE), vitality, proliferation, migration, the production of sulfated glycosaminoglycans (sGAG) and chondrogenic differentiation capacity were evaluated by histological staining (DAPI, Phalloidin, Live-Dead), biochemical assays (alamarBlue®, PicoGreen®, DMMB) and the quantification of gene expression (qPCR). Results show that cell vitality and CSE were not influenced by DPNC zones. ASCs, however, showed a significantly higher proliferation and elevated expression of early chondrogenic differentiation, as well as fibrocartilage markers, on the OS. On the contrary, there was a significantly higher upregulation of hypertrophy marker MMP13 (p < 0.0001) and GAG production (p = 0.0105) on the IS, whereas cell invasion into the three-dimensional DPNC was higher in comparison to the OS. We conclude that the zonal-dependent distinct architecture and composition of NC modulates activities of ASCs seeded on DPNC. These findings might be used for engineering of cartilage substitutes needed in facial reconstructive surgery that yield an equivalent histological and functional structure, such as native NC.

Evaluation of human nasal cartilage nonlinear and rate dependent mechanical properties.

Nasal reconstruction frequently requires donor cartilage and tissue, and ideally, donor tissue will closely emulate native nasal cartilage mechanics. Tissue engineering scaffolds, especially 3D printed scaffolds, have been proposed for nasal reconstruction, and the success of these constructs may depend on how well scaffolds reflect native nasal cartilage mechanical properties. Therefore, consistent and comprehensive characterization of native nasal cartilage mechanical properties is a foundation for nasal cartilage tissue engineering and reconstruction in general by providing design targets for reconstructive materials. Our group has previously shown the feasibility of producing scaffolds with porous architecture permitting chondrocyte growth and cartilage production. In this study, we determined the nonlinear and stress relaxation behavior of human nasal cartilage under unconfined compression. We then fit this experimental data to nonlinear elastic, nonlinear viscoelastic and nonlinear biphasic constitutive models. The resulting coefficients will provide design targets for nasal reconstruction and scaffold design as well as outcome measures for assessment of tissue engineered nasal cartilage.

A Novel Approach to Septal Perforation Repair: Septal Cartilage Cells Induce Chondrogenesis of hASCs In Vitro.

The aim of this study was to investigate the effect of medium harvested from septal cartilage cells on chondrogenic differentiation of adipose stem cells (hASCs) and to compare/contrast its properties to those of a commonly used standard medium formulation in terms of induction and maintenance of chondrogenic hASCs. Differentiation was carried out under three different conditions: septal cartilage medium-SCM, chondrogenic differentiation medium-CM, and 50:50 mixture of CM/SCM. Mesenchymal stem cells (MSCs) markers were determined by flow cytometry. The cytotoxic and apoptotic effects were determined by MTS and Annexin V assay, respectively. The differentiation status of the cells was confirmed by Alcian blue staining, and quantitative real-time flow cytometry showed that hASCs were positive for MSCs, negative for hematopoietic stem cells and endothelial cell surface markers. According to MTS analysis, the first condition was not toxic at any concentration tested. Annexin V assay revealed that the application of different concentrations of SCM did not result in any cell death. The Alcian blue and gene expression analyses showed that the cells in the SCM group underwent the highest cartilage cell formation. The observed increase in chondrogenesis may offer better treatment options for the cartilage defects seen in nasal septum perforation.

Combination of bioactive factors and IEIK13 self-assembling peptide hydrogel promotes cartilage matrix production by human nasal chondrocytes.

Nasal reconstruction remains a challenge for every reconstructive surgeon. Alloplastic implants are proposed to repair nasal cartilaginous defects but they are often associated with high rates of extrusion and infection and poor biocompatibility. In this context, a porous polymeric scaffold filled with an autologous cartilage gel would be advantageous. In this study, we evaluated the capacity of IEIK13 self-assembling peptide (SAP) to serve as support to form such cartilage gel. Human nasal chondrocytes (HNC) were first amplified with FGF-2 and insulin, and then redifferentiated in IEIK13 with BMP-2, insulin, and T3 (BIT). Our results demonstrate that IEIK13 fosters HNC growth and survival. HNC phenotype was assessed by RT-PCR analysis and neo-synthesized extracellular matrix was characterized by western blotting and immunohistochemistry analysis. BIT-treated cells embedded in IEIK13 displayed round morphology and expressed cartilage-specific markers such as type II and type IX collagens and aggrecan. In addition, we did not detect significant production of type I and type X collagens and gene products of dedifferentiated and hypertrophic chondrocytes that are unwanted in hyaline cartilage. The whole of these results indicates that the SAP IEIK13 represents a suitable support for hydrogel-based tissue engineering of nasal cartilage. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 893-903, 2019.

Signals from the brain and olfactory epithelium control shaping of the mammalian nasal capsule cartilage.

Facial shape is the basis for facial recognition and categorization. Facial features reflect the underlying geometry of the skeletal structures. Here, we reveal that cartilaginous nasal capsule (corresponding to upper jaw and face) is shaped by signals generated by neural structures: brain and olfactory epithelium. Brain-derived Sonic Hedgehog (SHH) enables the induction of nasal septum and posterior nasal capsule, whereas the formation of a capsule roof is controlled by signals from the olfactory epithelium. Unexpectedly, the cartilage of the nasal capsule turned out to be important for shaping membranous facial bones during development. This suggests that conserved neurosensory structures could benefit from protection and have evolved signals inducing cranial cartilages encasing them. Experiments with mutant mice revealed that the genomic regulatory regions controlling production of SHH in the nervous system contribute to facial cartilage morphogenesis, which might be a mechanism responsible for the adaptive evolution of animal faces and snouts.

Characteristics of Nasal Septal Cartilage-Derived Progenitor Cells during Prolonged Cultivation.

Objective To produce alternate cell sources for tissue regeneration, human nasal septal cartilage-derived progenitor cells (NSPs) were tested to identify whether these cells meet the criteria of cartilage progenitor cells. We also evaluated the effects of prolonged cultivation on the characteristics of NSPs. Study Design In vitro study. Setting Academic research laboratory. Methods NSPs were isolated from discarded human nasal septal cartilage. NSPs were cultured for 10 passages. The expression of septal progenitor cell surface markers was assessed by fluorescence-activated cell sorting. Cell proliferation was measured with a cell-counting kit. Cytokine secretion was analyzed with multiplex immunoassays. Chondrogenic differentiation of NSPs without differentiation induction was analyzed with type II collagen immunohistochemistry. Cartilage-specific protein expression was evaluated by Western blotting. Under osteo- and adipodifferentiation media, 2 lineage differentiation potentials were evaluated by histology and gene expression analysis. Results Surface epitope analysis revealed that NSPs are positive for mesenchymal stem cells markers and negative for hematopoietic cell markers. Cultured NSPs showed sufficient cell expansion and chondrogenic potential, as demonstrated by immunostaining and expression of cartilage-specific protein. IL-6, IL-8, and transforming growth factor ß were secreted by over 200 pg/mL. The osteo- and adipodifferentiation potentials of NSPs were identified by histology and specific gene expression. The aforementioned characteristics were not influenced by prolonged cultivation. Conclusion NSPs represent an initial step toward creating a cell source from surgically discarded tissue that may prove useful in cartilage regeneration.

In vitro extracellular matrix accumulation of nasal and articular chondrocytes for intervertebral disc repair.

Chondrocyte based regenerative therapies for intervertebral disc repair such as Autologous Disc Cell Transplantation (ADCT, CODON) and allogeneic juvenile chondrocyte implantation (NuQu®, ISTO Technologies) have demonstrated good outcomes in clinical trials. However concerns remain with the supply demand reconciliation and issues surrounding immunoreactivity which exist for allogeneic-type technologies. The use of stem cells is challenging due to high growth factor requirements, regulatory barriers and differentiation towards a stable phenotype. Therefore, there is a need to identify alternative non-disc cell sources for the development and clinical translation of next generation therapies for IVD regeneration. In this study, we compared Nasal Chondrocytes (NC) as a non-disc alternative chondrocyte source with Articular Chondrocytes (AC) in terms of cell yield, morphology, proliferation kinetics and ability to produce key extracellular matrix components under 5% and 20% oxygen conditions, with and without exogenous TGF-ß supplementation. Results indicated that NC maintained proliferative capacity with high amounts of sGAG and lower collagen accumulation in the absence of TGF-ß supplementation under 5% oxygen conditions. Importantly, osteogenesis and calcification was inhibited for NC when cultured in IVD-like microenvironmental conditions. The present study provides a rationale for the exploration of nasal chondrocytes as a promising, potent and clinically feasible autologous cell source for putative IVD repair strategies.

Generating Mechanically Stable, Pediatric, and Scaffold-Free Nasal Cartilage Constructs In Vitro.

Traditional methods of cartilage tissue engineering rely on the use of scaffolds. Although successful chondrogenesis has been reported in scaffold-based constructs, the use of exogenous materials has limited their application due to eliciting host immunogenic responses and potentially resulting in construct failure. As a result, tissue engineering approaches, which aim to generate scaffold-free cartilaginous constructs, have become of particular interest. Here, we generated stable three-dimensional scaffold-free cartilaginous constructs by cultivating expanded pediatric nasal chondrocyte multilayers in a slow turning lateral vessel bioreactor system under chemically defined media. Bioreactor cultivation resulted in increased construct cellularity, fourfold tissue thickness, and 200% sulfated glycosaminoglycan deposition with respect to static culture equivalent cultures. These improvements led to significantly enhanced mechanical and biochemical properties of bioreactor-cultivated constructs, allowing them to support their own weight, while static culture constructs remained fragile. Consequently, bioreactor-cultivated constructs closely resembled native nasal cartilage tissue histologically, mechanically, and biochemically. We propose that this method of cartilage construct formation could be used to obtain readily available human scaffold-free cartilaginous constructs.

Cartilage Regeneration in the Head and Neck Area: Combination of Ear or Nasal Chondrocytes and Mesenchymal Stem Cells Improves Cartilage Production.

BACKGROUND: Cartilage tissue engineering can offer promising solutions for restoring cartilage defects in the head and neck area and has the potential to overcome limitations of current treatments. However, to generate a construct of reasonable size, large numbers of chondrocytes are required, which limits its current applicability. Therefore, the authors evaluate the suitability of a combination of cells for cartilage regeneration: bone marrow-derived mesenchymal stem cells and ear or nasal chondrocytes. METHODS: Human bone marrow-derived mesenchymal stem cells were encapsulated in alginate hydrogel as single-cell-type populations or in combination with bovine ear chondrocytes or nasal chondrocytes at an 80:20 ratio. Constructs were either cultured in vitro or implanted directly subcutaneously into mice. Cartilage formation was evaluated with biochemical and biomechanical analyses. The use of a xenogeneic coculture system enabled the analyses of the contribution of the individual cell types using species-specific gene-expression analyses. RESULTS: In vivo, human bone marrow-derived mesenchymal stem cells/bovine ear chondrocytes or human bone marrow-derived mesenchymal stem cells/bovine nasal chondrocytes contained amounts of cartilage components similar to those of constructs containing chondrocytes only (i.e., bovine ear and nasal chondrocytes). In vitro, species-specific gene-expression analyses demonstrated that aggrecan was expressed by the chondrocytes only, which suggests a more trophic role for human bone marrow-derived mesenchymal stem cells. Furthermore, the additional effect of human bone marrow-derived mesenchymal stem cells was more pronounced in combination with bovine nasal chondrocytes. CONCLUSIONS: By supplementing low numbers of bovine ear or nasal chondrocytes with human bone marrow-derived mesenchymal stem cells, the authors were able to engineer cartilage constructs with properties similar to those of constructs containing chondrocytes only. This makes the procedure more feasible for future applicability in the reconstruction of cartilage defects in the head and neck area because fewer chondrocytes are required.

Poly(γ-Glutamic Acid) as an Exogenous Promoter of Chondrogenic Differentiation of Human Mesenchymal Stem/Stromal Cells.

Cartilage damage and/or aging effects can cause constant pain, which limits the patient's quality of life. Although different strategies have been proposed to enhance the limited regenerative capacity of cartilage tissue, the full production of native and functional cartilaginous extracellular matrix (ECM) has not yet been achieved. Poly(γ-glutamic acid) (γ-PGA), a naturally occurring polyamino acid, biodegradable into glutamate residues, has been explored for tissue regeneration. In this work, γ-PGA's ability to support the production of cartilaginous ECM by human bone marrow mesenchymal stem/stromal cells (MSCs) and nasal chondrocytes (NCs) was investigated. MSC and NC pellets were cultured in basal medium (BM), chondrogenic medium (CM), and CM-γ-PGA-supplemented medium (CM+γ-PGA) over a period of 21 days. Pellet size/shape was monitored with time. At 14 and 21 days of culture, the presence of sulfated glycosaminoglycans (sGAGs), type II collagen (Col II), Sox-9, aggrecan, type XI collagen (Col XI), type X collagen (Col X), calcium deposits, and type I collagen (Col I) was analyzed. After excluding γ-PGA's cytotoxicity, earlier cell condensation, higher sGAG content, Col II, Sox-9 (day 14), aggrecan, and Col X (day 14) production was observed in γ-PGA-supplemented MSC cultures, with no signs of mineralization or Col I. These effects were not evident with NCs. However, Sox-9 (at day 14) and Col X (at days 14 and 21) were increased, decreased, or absent, respectively. Overall, γ-PGA improved chondrogenic differentiation of MSCs, increasing ECM production earlier in culture. It is proposed that γ-PGA incorporation in novel biomaterials has a beneficial impact on future approaches for cartilage regeneration.

Cellular proliferation in the nasal septal cartilage of juvenile minipigs.

The growth of the nasal septal cartilage is believed to be a driving force of midfacial growth. Cellular proliferation is an important contributor to growth of the cartilage, but this factor has been rarely investigated. The current study was undertaken to assess the proliferation and cellular density in the septal cartilage of fast-growing juvenile minipigs. Six minipigs averaging 4.4 ±â€…1 months old were injected with 5'-bromo-2'-deoxyuridine (BrdU), a thymidine analog, 24 h before death. The septal cartilage was sectioned in the coronal plane and reacted for BrdU. The proliferative index (number of BrdU-positive chondrocytes/total number of chondrocytes) and cellular density (number of cells mm(-2) ) of various locations of the septum were measured and compared in order to determine overall proliferation rate and whether regional variations in proliferative activity and cellular density are present. To provide a time perspective to the problem of midfacial growth, the lengths of the nasal bone and the palate were measured in a collection of 61 dry skulls of minipigs aged 1-8 months. Results showed that the septal chondrocytes were proliferating at a surprisingly high rate (~21%). The proliferative index was higher in the ventral and middle compared with the dorsal locations, and in the central cartilage compared with the perichondrium. No difference in proliferative index was found between the anterior and posterior parts of the septum. Cellular density was higher in the perichondrium than in the central cartilage. Within the central cartilage there was a trend for higher cellular density anteriorly. In conclusion, the rapidly growing midface of juvenile minipigs is associated with a high rate of septal proliferation, especially in the ventral half of the cartilage.

Tissue-engineered cartilage for facial plastic surgery.

PURPOSE OF REVIEW: The reconstruction of cartilaginous craniofacial defects is ideally performed with analogous grafting material, such as autologous tissue. However, the use of autologous cartilage is limited by its finite availability and potentially suboptimal geometry to repair specific defects. Tissue engineering of human cartilage may provide the adequate supply of grafting and implant material for the reconstruction of cartilaginous facial defects. An update of the various cartilage tissue engineering methodologies is provided in this review. RECENT FINDINGS: The cartilage tissue engineering paradigm begins with the harvest of a small septal cartilage donor specimen. This is followed by the isolation and subsequent proliferation of chondrocytes and the seeding of these cells onto three-dimensional scaffolds. Neocartilage is created as pericellular substrate, is produced by the cells and deposited throughout the scaffold. Theoretically, the mature cartilage construct can be introduced back into the same patient for reconstruction of craniofacial defects. Initial steps of the cartilage tissue engineering protocol have been standardized; however, modifications of subsequent steps have shown the potential to profoundly impact tissue composition and strength, bringing the properties of cartilage constructs closer to those of native human septum. SUMMARY: The ability to engineer virtually limitless quantities of autologous cartilage could have a profound impact on facial plastic and reconstructive surgery. The strategies used to refine human cartilage culture techniques have successfully produced neocartilage constructs with biochemical and biomechanical properties approaching those of native septal tissue. With the steady progress achieved in recent years, there is great capacity for the proximate realization of surgically implantable tissue-engineered cartilage constructs.

Engineered nasal cartilage by cell homing: a model for augmentative and reconstructive rhinoplasty.

BACKGROUND: Current augmentative and reconstructive rhinoplasties use auto logous tissue grafts or synthetic bioinert materials to repair nasal trauma or attain an aesthetic shape. Autologous grafts are associated with donor-site trauma and morbidity. Synthetic materials are widely used but often yield an unnatural appearance and are prone to infection or dislocation. There is an acute clinical need for the generation of native tissues to serve as rhinoplasty grafts without the undesirable features that are associated with autologous grafts or current synthetic materials. METHODS: Bioactive scaffolds were developed that not only recruited cells in the nasal dorsum in vivo, but also induced chondrogenesis of the recruited cells. Bilayered scaffolds were fabricated with alginate-containing gelatin microspheres encapsulating cytokines atop a porous poly(lactic-co-glycolic acid) base. Microspheres were fabricated to contain recombinant human transforming growth factor-ß3 at doses of 200, 500, or 1000 ng, with phosphate-buffered saline-loaded microspheres used as a control. A rat model of augmentation rhinoplasty was created by implanting scaffolds atop the native nasal cartilage surface that was scored to induce cell migration. Tissue formation and chondrogenesis in the scaffolds were evaluated by image analysis and histologic staining with hematoxylin and eosin, toluidine blue, Verhoeff elastic-van Geison, and aggrecan immunohistochemistry. RESULTS: Sustained release of increasing doses of transforming growth factor-ß3 for up to the tested 10 weeks promoted orthotopic cartilage-like tissue formation in a dose-dependent manner. CONCLUSIONS: These findings represent the first attempt to engineer cartilage tissue by cell homing for rhinoplasty, and could potentially serve as an alternative material for augmentative and reconstructive rhinoplasty.

Marine collagen scaffolds for nasal cartilage repair: prevention of nasal septal perforations in a new orthotopic rat model using tissue engineering techniques.

Autologous grafts are frequently needed for nasal septum reconstruction. Because they are only available in limited amounts, there is a need for new cartilage replacement strategies. Tissue engineering based on the use of autologous chondrocytes and resorbable matrices might be a suitable option. So far, an optimal material for nasal septum reconstruction has not been identified. The aim of our study was to provide the first evaluation of marine collagen for use in nasal cartilage repair. First, we studied the suitability of marine collagen as a cartilage replacement matrix in the context of in vitro three dimensional cultures by analyzing cell migration, cytotoxicity, and extracellular matrix formation using human and rat nasal septal chondrocytes. Second, we worked toward developing a suitable orthotopic animal model for nasal septum repair, while simultaneously evaluating the biocompatibility of marine collagen. Seeded and unseeded scaffolds were transplanted into nasal septum defects in an orthotopic rat model for 1, 4, and 12 weeks. Explanted scaffolds were histologically and immunohistochemically evaluated. Scaffolds did not induce any cytotoxic reactions in vitro. Chondrocytes were able to adhere to marine collagen and produce cartilaginous matrix proteins, such as collagen type II. Treating septal cartilage defects in vivo with seeded and unseeded scaffolds led to a significant reduction in the number of nasal septum perforations compared to no replacement. In summary, we demonstrated that marine collagen matrices provide excellent properties for cartilage tissue engineering. Marine collagen scaffolds are able to prevent septal perforations in an autologous, orthotopic rat model. This newly described experimental surgical procedure is a suitable way to evaluate new scaffold materials for their applicability in the context of nasal cartilage repair.

Protective effects of polydeoxyribonucleotides on cartilage degradation in experimental cultures.

The capacity of cartilage self-regeneration is considered to be limited. Joint injuries often evolve in the development of chronic wounds on the cartilage surface. Such lesions are associated with articular cartilage degeneration and osteoarthritis. Re-establishing a correct micro/macro-environment into damaged joints could stop or prevent the degenerative processes. This study investigated the effect of polydeoxyribonucleotides (PDRNs) on cartilage degradation in vitro and on cartilage extracted cells. The activities of matrix metalloproteinases 2 and 9 were measured in PDRN-treated cells and in controls at days 0 and 30 of culture. Human nasal cartilage explants were cultured, and the degree of proteoglycan degradation was assessed by measuring the amount of glycosaminoglycans released into the culture medium. The PDRN properties compared with controls were tested on cartilage tissues to evaluate deposition of extracellular matrix. Chondrocytes treated with PDRNs showed a physiological deposition of extracellular matrix (aggrecan and type II collagen: Western blot, IFA, fluorescence activated cell sorting, Alcian blue and safranin O staining). PDRNs were able to inhibit proteoglycan degradation in cartilage explants. The activities of matrix metalloproteinases 2 and 9 were reduced in all PDRN-treated samples. Our results indicate that PDRNs are suitable for a long-term cultivation of in vitro cartilage and have therapeutic effects on chondrocytes by protecting cartilage.

Effects of different levels of crushing on the viability of rabbit costal and nasal septal cartilages.

BACKGROUND: Cartilage grafts are frequently used in nasal surgery for structural and/or aesthetic purposes. The literature holds contradictory reports concerning the effect of crushing on the viability of cartilage grafts. METHODS: Nasal septal and costal cartilage grafts were harvested from 12 New Zealand rabbits. Each nasal septal and costal cartilage was divided into five equal pieces. One of the pieces was left intact and the remaining four were prepared as slightly, moderately, severely, or significantly crushed. The cartilage pieces were then autoimplanted into the paravertebral skin of the rabbits. The animals were euthanized 4 months later and the effect of crushing on cartilage grafts was assessed pathologically. RESULTS: The viability of the chondrocytes was found to be decreased as the level of crushing increased. The mean chondrocyte viability rates for the intact, slightly crushed, moderately crushed, severely crushed, and significantly crushed cartilages were 88, 75, 51, 41, and 13 percent for the septal cartilages and 94, 83, 62, 32, and 26 percent for the costal cartilages, respectively. The differences between the mean viability rates of septal and costal cartilage groups were statistically not significant. CONCLUSIONS: The level of crushing determines the rate of viability for the crushed cartilage. Viability rates and the clinical properties of the slightly crushed cartilage grafts at long-term follow-up may be similar to those of the intact cartilage grafts. However, severe or significant crushing leads to a decrement in the viability of the chondrocytes and may cause unpredictable degrees of volume loss at long-term follow-up.

Septal cartilage tissue engineering: new horizons.

Cartilage tissue engineering is a dynamically changing field that has the potential to address some of the tissue repair challenges seen in nasal and craniofacial reconstructive surgeries. The scope of the problem includes limited autologous tissue availability, donor site morbidity associated with the harvesting of these tissue grafts, and the risk of an immune reaction to allogenic or synthetic implants that might be used as alternatives. Current tissue engineering strategies involve harvesting a small biopsy specimen from a patient and then isolating chondrocytes through enzymatic digestion of the extracellular matrix. These isolated chondrocytes can be expanded in monolayer and reseeded into a three-dimensional scaffold that could potentially be used as autologous surgical grafts. Using cell-expansion techniques, it would be feasible to generate abundant amounts of cartilage in defined shapes and sizes. The ideal tissue-engineered cartilage would resemble native tissue in terms of its biochemical, structural, and metabolic properties so that it could restore stability, function, and contour to the damaged or defective facial region. In this article, emerging technology and major challenges are described to highlight recent advances and overall trends within septal cartilage tissue engineering.

Type II and VI collagen in nasal and articular cartilage and the effect of IL-1alpha on the distribution of these collagens.

The distribution of type II and VI collagen was immunocytochemically investigated in bovine articular and nasal cartilage. Cartilage explants were used either fresh or cultured for up to 4 weeks with or without interleukin 1alpha (IL-1alpha). Sections of the explants were incubated with antibodies for both types of collagen. Microscopic analyses revealed that type II collagen was preferentially localized in the interchondron matrix whereas type VI collagen was primarily found in the direct vicinity of the chondrocytes. Treatment of the sections with hyaluronidase greatly enhanced the signal for both types of collagen. Also in sections of explants cultured with IL-1alpha a higher level of labeling of the collagens was found. This was apparent without any pre-treatment with hyaluronidase. Under the influence of IL-1alpha the area positive for type VI collagen that surrounded the chondrocytes broadened. Although the two collagens in both types of cartilage were distributed similarly, a remarkable difference was the higher degree of staining of type VI collagen in articular cartilage. Concomitantly we noted that digestion of this type of cartilage hardly occurred in the presence of IL-1alpha whereas nasal cartilage was almost completely degraded within 18 days of culture. Since type VI collagen is known to be relatively resistant to proteolysis we speculate that the higher level of type VI collagen in articular cartilage is important in protecting cartilage from digestion.

[Chondroblast proliferative activity study in forming big alar cartilage].

Study was performed of chondroblast proliferative activity peculiarities on early stages of big alar cartilage forming. The reaction with monoclonal antibodies towards proliferating cell nuclear antigen in alar cartilage of human embryo and newly born infant rat testified to 2 cartilage growth types - appositional and interstitial. Cells proliferative activity did not depend upon their location on inner or outer side of nasal cartilaginous plate thereby not conforming existent in the literature opinion about the presence of special zones of cartilaginous tissue primary new formation in human nasal cartilage on early stages of its ontogeny.