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Tetracycline impregnation delays collagen membrane degradation in vivo.

BACKGROUND: Guided tissue and bone regeneration using bioabsorbable collagen membranes is a common practice. Collagen promotes progenitor cell adhesion, chemotaxis, homeostasis, and physiologic degradation with low immunogenicity, which makes it an ideal material for barrier preparation. Collagen membranes have to maintain integrity for a proper time, thus ensuring successful cell exclusion. Early collagen membrane degradation is detrimental for the success of regenerative procedures. This in vivo study was conducted to evaluate the effect of soaking collagen membranes in different tetracycline hydrochloride (TCN) concentration solutions on its degradation. METHODS: Five mm disks of collagen membrane were soaked in either 100 mg/ml TCN (group 100) or 50 mg/ml TCN (group 50); a group of non-treated disks served as controls. All disks were labeled with aminohexanoyl-biotin-N-hydroxy-succinimide ester (biotin) and implanted in rat calvaria bone. Block sections were taken after 3 weeks and histological slides stained with horseradish peroxidase (HRP) to detect remnants of biotinylated collagen. Staining intensity was analyzed by image-analysis software taking quadruplicate measurements of a 500 microm2 area each. Data were analyzed using analysis of variance (ANOVA) with repeated measures and paired t test with Bonferroni correction. RESULTS: Staining intensity of membranes in group 100 was > 5-fold higher than the control while group 50 exhibited > 11-fold higher intensity than the control and > 2.5-fold higher than the 100. All of these differences were statistically significant (P < 0.001). CONCLUSION: Soaking collagen membranes in 50 mg/ml TCN solution is a useful, practical, and simple tool to slow membrane degradation.

Absorbable Implants↗

MR imaging in biofix-osteosynthesis.

PURPOSE: Osteosynthesis by means of bioresorbable implants, mostly of self-reinforced poly-L-lactide (SR-PLLA), has been used in humans for about 10 years. The aim of this study was to examine the controversy between histological studies confirming fragmentation of the biomaterial and radiological studies showing no breaking of the material. MATERIAL AND METHODS: Six patients with displaced malleolar fractures operatively treated with biodegradable SR-PLLA screws underwent MR examinations at 1.5 T, immediately postoperatively and after one to two years. RESULTS: The biodegradable osteosynthetic screws were clearly seen on all MR images. Of 12 screws, 6 were broken at the final examination (5 syndesmotic transfixation screws and 1 screw through the growth cartilage). CONCLUSION: The breaking of a biodegradable osteosynthesis is possible to document on MR images.

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Experimental study on tissue engineering of the small intestine by mesenchymal stem cell seeding.

BACKGROUND: We have succeeded in regenerating the small intestine by the use of tissue engineering techniques. However, the regenerated intestine proved to lack the muscle layer, which is essential for functional peristalsis. To induce regeneration of the muscle layer, we focused on autologous mesenchymal stem cells (MSC) as a source of muscle tissue. The aim of this study was to investigate the effect of MSC seeding onto the collagen scaffold on induction of the muscle layer. MATERIALS AND METHODS: We used six female beagle dogs. The small intestine was resected over a length of 5 cm and reconstructed by a collagen sponge graft in the same way as in our previous study. Autologous MSC derived from bone marrow (10(7) cells) were seeded onto the collagen sponge just before implantation. Animals were sacrificed at 2, 4, and 16 weeks after surgery, and specimens were examined histologically. RESULTS: All six dogs survived until the scheduled time of sacrifice. At 4 weeks, regeneration of the intestine was observed at the reconstructed site. Cells positive for alpha-smooth muscle actin appeared on the scaffold in the MSC-seeded group. However, they disappeared by 16 weeks and only a thin muscle layer regenerated beneath the mucosal layer, although the regenerated mucosal layer covered the luminal surface of the regenerated intestine. CONCLUSIONS: MSC seeding induced a transient distribution of cells positive for alpha-smooth muscle actin on the scaffold, but did not induce regeneration of the muscle layer. Further investigation is necessary to achieve this aim.

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In vivo tissue response to resorbable silica xerogels as controlled-release materials.

Biodegradable, controlled-release carrier materials with non-toxic degradation products are valuable for local delivery of biologically active molecules. Previously, it was shown that room-temperature processed silica sol-gels (or xerogels) are porous, resorbable materials that can release molecules of various sizes in a controlled, time dependent manner. Previous in vitro studies also demonstrated benefits of silica xerogels as controlled-release materials for the treatment of bone infections. Herein the tissue and cell response to xerogels is documented using a subacute implantation procedure. The tissue response was correlated to composition, surface properties, resorption rate and incorporation of the antibiotic vancomycin. Ca- and P-free and Ca- and P-containing xerogels, with and without apatite (AP) surface, were used. Xerogels were implanted either as discs in a subcutaneous site, or as granules in the iliac crest of New Zealand white rabbits. The samples with surrounding tissue were retrieved after 2 and 4 weeks of implantation. Silica xerogels implanted either as discs subcutaneously or as granules in the iliac crest showed a favorable tissue response. The granules, either with or without Ca and P content, gradually resorbed over time. The resorption was accompanied by extensive trabecular bone growth and a minimal inflammatory response. Ca- and P-containing granules with an AP-surface layer showed a slower resorption rate and more extensive new bone growth than those without AP layer. Among AP-coated granules, those with incorporated vancomycin showed the most favorable tissue response. The present in vivo data together with prior in vitro data suggest that these xerogels have potential as controlled-release materials for the treatment of bone infections and as carrier materials for a variety of other applications.

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Use of lyodura for bone augmentation of osseous defects around dental implants.

BACKGROUND: Lyodura has been used in periodontology and maxillofacial surgery to overcome different clinical conditions. The use of lyodura to induce new bone formation in humans has not been widely reported. The purpose of this article is to describe the use of lyodura as a resorbable barrier to promote bone formation in osseous defects around dental implants. METHODS: The study group consisted of 22 healthy patients (12 women and 10 men), with a mean age of 32 (range 20 to 45). A total of 44 implants (9 titanium screw-type and 35 hydroxyapatite-coated cylinder implants) were placed. Of these, 27 implants were placed in the maxilla and 17 in the mandible. At 27 extraction sites bony vertical defects were measured on the buccal, lingual, mesial and distal sides of the implant. In the remaining 17 cases, where either immediate or staged implantation was performed, and a dehiscence was present, its vertical length was measured from the alveolar crest. All bony defects were grafted with autogenous bone harvested from the tuberosity. Each site was covered with lyodura. RESULTS: Healing was uneventful; no inflammation, infection, or soft tissue dehiscence was observed. At all extraction sites and in 76% of the dehiscence defects, hard tissue appearing clinically similar to bone completely filled the defect. Of those defects not completely filled, a mean vertical bone increase of 2.5 mm was recorded. A mean vertical bone increase of 2.6 mm was achieved at extraction sites and a mean increase of 2.79 mm was achieved at dehiscence sites. CONCLUSIONS: Lyodura can safely and effectively be used for guided bone regeneration at both extraction sites and for dehiscence defects.

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[Repair of cranial defects with bone marrow derived mesenchymal stem cells and beta-TCP scaffold in rabbits].

OBJECTIVE: To determine whether culture expanded bone marrow derived mesenchymal stem cells (MSCs) in combination with beta-tricalcium phosphate(beta-TCP) can repair critical cranial defects in New Zealand rabbits. METHODS: In group A(n = 20), MSCs from homogeneous rabbits were isolated and expanded in vitro and then implanted onto the pre-molded porous beta-TCP. The MSCs-beta-TCP complexes were implanted into rabbit critical cranial defects. In group B (n = 10), The defects were repaired with beta-TCP only. In group C(n = 4), the defects were left un-repaired. Samples were extracted 6 and 12 weeks after operation for histological, histochemical and immunohistochemical analysis. RESULTS: In group A, bone-like tissue formation could be seen on the surface of the implants. Microscopic analysis demonstrated certain degradation of beta-TCP and extensive new bone filling in rich extracellular matrix after 6 weeks. The cells were stained positively for type I collagen. After 12 weeks, the bioceramics had almost completely degraded and abundant bone formation could be seen in the whole defects. In group B, marginal bone ingrowth was observed after 6 weeks and the number of osteoblasts increased significantly after 12 weeks. However, no new bone formation could be detected in the middle of the material. In group C, only a small quantity of new bone formation was found along the margin of defects. CONCLUSION: Transplantation of MSCs with beta-TCP can serve as an example of a cell-based treatment for bone regeneration in skeletal defects.

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Asian experience with the Pintucci keratoprosthesis.

PURPOSE: To report the outcome of a retrospective study of the Pintucci Biointegrable Keratoprosthesis (PBIKP) in Asian eyes. MATERIALS AND METHODS: This was a retrospective analysis of 31 eyes of 31 consecutive patients, who underwent surgery with implantation of the PBIKP. The age ranged from 7 years to 65 years, with a mean age of 34 years. The indications for the PBIKP were chemical burns (11 cases); highly vascularized failed grafts (11 cases); severe dry eyes with total vascularized cornea (6 cases); miscellaneous (3 cases). Twenty eight bilaterally blind Asian patients, with vision not exceeding hand motion close to face in the better eye, underwent the classical two-stage procedure to implant the PBIKP. In another three patients, the PBIKP was implanted as a one-stage procedure. All 31 eyes were unsuitable for keratoplasty or had repeated failed keratoplasties. All patients were followed up for periods ranging from 6 months to 7 years, with a mean of 3.2 years. RESULTS: No eye had infection or retro prosthetic membrane, which were common complications in other Keratoprostheses designs. Twenty-four of 31 eyes improved to greater than finger counting at 1.5 metres, enabling the patients to function independently. Four of 31 eyes (13%) improved to 20/200 or better. Twelve of 31 eyes had significant complications. However only a few were vision- threatening. CONCLUSION: With careful patient selection, the PBIKP could prove to be a useful modality of treatment for bilaterally corneally blind Asian patients, whose vision cannot be improved with conventional medical or surgical treatments.

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A long-term clinical study on dislocated ankle fractures fixed with self-reinforced polylevolactide (SR-PLLA) implants.

Sixteen patients with dislocated ankle fractures fixed between 1988 and 1991 with self-reinforced poly(L-lactide; SR-PLLA) screws and/or rods were followed up after 8.6 to 11.7 years (mean 9.6 years) at the Department of Orthopaedics and Traumatology, Helsinki University Central Hospital. In all patients accurate reduction of the fractures was retained and uneventful bony union was achieved. Good or excellent long-term functional results were observed in 15 out of 16 patients. One patient had post-traumatic osteoarthritis. In 5 patients, a late tissue reaction was observed over an extruding screw head with mild symptoms, which led to removal of small palpable masses. There were two superficial wound infections, one after a primary operation and one caused by a late tissue reaction after an operation. The correct operative technique, where all extruding extraosseous SR-PLLA material should be removed during the primary operation, should be followed.

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Mats made from fibronectin support oriented growth of axons in the damaged spinal cord of the adult rat.

A variety of biological as well as synthetic implants have been used to attempt to promote regeneration into the damaged spinal cord. We have implanted mats made from fibronectin (FN) into the damaged spinal cord to determine their effectiveness as a substrate for regeneration of axons. These mats contain oriented pores and can take up and release growth factors. Lesion cavities 1 mm in width and depth and 2 mm in length were created on one side of the spinal cord of adult rats. FN mats containing neurotrophins or saline were placed into the lesion. Mats were well integrated into surrounding tissue and showed robust well-oriented growth of calcitonin gene-related peptide, substance P, GABAergic, cholinergic, glutamatergic, and noradrenergic axons into FN mats. Transganglionic tracing using cholera toxin B indicated large-diameter primary afferents had grown into FN implants. Schwann cells had also infiltrated FN mats. Electron microscopy confirmed the presence of axons within implants sites, with most axons either ensheathed or myelinated by Schwann cells. Mats incubated in brain-derived neurotrophic factor and neurotrophin-3 showed significantly more neurofilament-positive and glutamatergic fibers compared to saline- and nerve growth factor-incubated mats, while mats incubated with nerve growth factor showed more calcitonin gene-related peptide-positive axons. In contrast, neurotrophin treatment had no effect on PGP 9.5-positive axons. In addition, in some animals with neurotrophin-3-incubated mats, cholera toxin B-labelled fibers had grown from the mat into adjoining intact areas of spinal cord. The results indicate that FN mats provide a substrate that is permissive for robust oriented axonal growth in the damaged spinal cord, and that this growth is supported by Schwann cells.

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Use of hydroxylapatite/ polymer-composite in facial bone augmentation. An experimental study.

On purpose to achieve better and more controlled alveolar ridge augmentation, biodegradable polyglycolide (PGA) curved implants with porous, particulate hydroxylapatite (HA) were developed to be used in an experimental augmentation in sheep mandible. Prior to surgery curved PGA implants were loaded with particulate HA with help of a rather fast degrading adhesive, polyglycolide/polylactide (PGA/PLA) copolymer. This augmentation device was inserted into the ridge using a slight modification of the tunnelling technique. Macroscopically, a dehiscence with loss of HA particles was observed in 3 of the 16 augmentations. Bone ingrowth was seen in a noticeable degree in one sheep at 24 weeks. Instead, foreign body-type cells were shown at the interface of mandibular bone and hydroxylapatite deposit, as well as abundant connective tissue reaction inside HA deposits. The results of the study give rise to concern about bony integration in the presence of biodegradable polyglycolide substances in HA augmentation. The findings call in question even their use as a carrier for bone forming agents in combination with HA.

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Comparison of the pull-out forces of bioabsorbable polylactide/glycolide screws (Biosorb and Lactosorb) and tacks: a study on the stability of fixation in human cadaver parietal bones.

The aim of this study was to compare the pull-out forces of bioabsorbable polylactide/glycolide (PLGA) tacks and screws in human cadaver parietal bones. Parietal bone pieces (c. 6 cm x 20 cm) were collected from five human male cadavers (age range: 47-75 years). Forty-nine BioSorbPDX (self-reinforced [SR] PLGA 80/20) tacks (1.5-mm diameter, 4.0-mm length), 47 BioSorbPDX (SR-PLGA 80/20) screws (1.5-mm diameter, 4.0-mm length), and 46 LactoSorb (PLGA 82/18) screws (1.5-mm diameter, 4.0-mm length) were applied. The tacks were applied to drill holes using a special applicator gun (no tapping or tightening). The screws were applied to drill holes in the traditional way using tapping and tightening with a screwdriver. A tensile testing machine was used. All the implants were tested thus: the head of the implant was held by an aluminum jig, and the jig was pulled with wire until implant failure. The testing pull speed was 10 mm/min. Means and SDs were calculated, and the data were analyzed using ANOVA. The pull-out force of the tacks was 115.9 +/- 8.3 N, that of Lactosorb screws was 112.9 +/- 12.1 N, and that of Biosorb screws was 110.4 +/- 8.9 N (statistically insignificant difference between the three groups). The most common reason for failure in the case of tacks was barb breakage (55.1%); it was thread breakage in the case of BioSorb screws (66%) and stem split in the case of Lactosorb screws (56%). Tacks seem to have a similar, perhaps even a little better, holding power to cranial bone as screws and can hence be recommended for clinical application, as the procedure saves time and, consequently, costs.

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Resorbable screws for fixation of autologous bone grafts.

The aim of this study was to evaluate the suitability of resorbable screws made of poly (D.L-lactide) acid (PDLLA) for fixation of autologous bone grafts related to graft regeneration and osseointegration of dental implants. In eight edentulous patients suffering from insufficient retention of their upper denture related to a severely resorbed maxilla, the floor of both maxillary sinus and width of the alveolar crest were augmented with an autologous bone graft from the iliac crest. Randomly, the bone graft used to augment the alveolar crest was fixed with two titanium screws on one side and two resorbable screws on the other side (split-mouth design). Three months after the reconstruction, a bone biopsy was taken with a trephine including one resorbable screw (N = 8). Subsequently, six implants were placed in the left and right posterior maxilla. Six months later, at the abutment connection, a bone biopsy was taken including the other resorbable screw (N = 8). The biopsies were processed for light microscopic examination. In addition, clinical parameters were scored. Wound healing was uneventful. Clinically no difference in wound healing was observed between sides treated with either a resorbable or titanium screw. No implants were lost. Six months after implantation, implant retained overdentures could be fabricated in all patients. All patients functioned well with their overdentures (follow-up 22.2 +/- 4.3 months). Three as well as 9 months after insertion (remnants of), the resorbable screws were still visible after reflecting the mucoperiosteum. Histological examination confirmed that a considerable quantity of remnants of the resorbable screws was still present, although areas with some fragmentation of the PDLLA were also observed. The screws were separated by a fibrous tissue layer containing many giant cells from the bone. Particles of PDLLA were observed within these giant cells. This study revealed that resorbable screws made of PDLLA can be used for fixation of bone grafts. The bulk of the PDLLA material is still present after 9 months.

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Inhibition of neointima formation after experimental coronary artery stenting: a new biodegradable stent coating releasing hirudin and the prostacyclin analogue iloprost.

BACKGROUND: To minimize acute stent thrombosis and development of restenosis, stents coated with biodegradable and nonbiodegradable polymers have been proposed to serve as sustained-release drug carriers. METHODS AND RESULTS: In both a sheep and a pig model, we examined the vascular response to standard and high-pressure implantation of coronary Palmaz-Schatz stents coated with a 10-microm layer of polylactic acid (MW 30 kDa) releasing recombinant polyethylene glycol (r-PEG)-hirudin and the prostacyclin analogue iloprost, both drugs with antithrombotic and potentially antiproliferative effects. Study observation time was 28 days. Between the corresponding stent groups, no differences were observed with regard to preplacement and postplacement implantation parameters. The morphometric analysis demonstrated that the coating was associated with a greater lumen diameter through a reduction in the mean restenosis area by 22.9% (P<0.02) in the standard-pressure model (sheep) and by 24.8% (P<0.02) in the overstretch pig model compared with uncoated control stents without inducing a local inflammatory response. CONCLUSIONS: The results from this study demonstrate beneficial effects of a polymeric stent coating with polylactic acid releasing r-PEG-hirudin and iloprost on the development of restenosis after coronary stent placement at 4 weeks, independent of the extent of vascular injury. Future studies are proposed to investigate the integration of other substances to further enhance the potential of the stent coating on reducing neointimal formation.

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Resorbable plate osteosynthesis of dislocated or pathological mandibular fractures: a prospective clinical trial of two amorphous L-/DL-lactide copolymer 2-mm miniplate systems.

The purpose of this study was to evaluate the indication for resorbable miniplates in traumatic and pathological mandibular fractures. Two resorbable miniplate systems, the 2.0-mm MacroSorb (Macropore, San Diego, Calif.) and the PolyMax (Synthes, Oberdorf, Switzerland), were prospectively used in 24 mandibular osteosyntheses. Made from amorphous 70:30 poly-L/DL-lactide, amorphous copolymer plates have not yet been evaluated for mandibular osteosyntheses. The main advantage of the amorphous copolymer-structure is continuous hydrolysis through water penetration into the implant body during the first 6 months. Hydrolysis breaks the copolymer chains into smaller particles that later become degraded through phagocytotic cells. Twelve patients, aged 13 to 83 years, were treated after providing informed consent. Fourteen dentate patients with moderately dislocated traumatic fractures and two edentulous atrophic and dislocated traumatic mandibular fractures were treated. Two patients with pathological fractures due to osteomyelitis received osteosynthesis after sequestrectomy. Histological specimens of the plates, screws, and surrounding soft tissues were taken after 3, 6, 9, and 12 months in secondary dental implant operations. A total of 22 osteosyntheses healed well without clinical or radiological signs of dislocation, insufficient or excess formation of callus, pseudarthrosis, or plate fracture. The follow-up time ranged from 4 to 22 months. One patient with osteomyelitis worsened because of widespread osseous infection, and one with atrophic fracture developed a bland fibrous pseudarthrosis. The histological specimens showed a moderate inflammatory foreign body reaction. No sinuous drainage or clinically apparent inflammation occurred. The presented osteosynthesis systems showed reliable stability for mandibular osteosynthesis in cooperative patients; however, two treatment failures occurred (8 percent). Disadvantages of the resorbable osteosyntheses were costs, greater diameter, screw breakage, and the need to place the screws vertically to the plate. The use of resorbable osteosyntheses in dislocated fractures should be further evaluated in controlled studies.

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Cartilage formation by serial passaged cultured chondrocytes in a new scaffold: hybrid 75:25 poly(L-lactide-epsilon-caprolactone) sponge.

PURPOSE: This study was designed to determine whether multipled chondrocytes immersed in a new scaffold, 75:25 poly(L-lactide-epsilon-caprolactone) sponge coated with type I collagen (75-PLC scaffold), could be used to generate cartilage tissue in vivo and to evaluate the correlation between cartilage generation and the phenotype of the proliferated chondrocytes. MATERIALS AND METHODS: Rat chondrocytes were suspended in 75-PLC scaffold at a density of 1 x 10 7 cells/mL after proliferation in a monolayer for 1 (P1) to 4 passages (P4) and implanted in nude mice for 4 weeks. Cells were characterized by the expression of genes encoding type II collagen, aggrecan, and type I collagen by Northern hybridization, and consequently, the newly formed tissue was evaluated histologically. RESULTS: The expression of aggrecan messenger RNA gradually decreased with the passaged cultures; however, the expression of type I collagen messenger RNA increased with time. The cartilage formations in all specimens were found not only in P1 chondrocytes but also in P2 chondrocytes, although when P3 chondrocytes were grafted, approximately 50% of cartilage formation was still observed up to but not beyond P4. CONCLUSION: It is suggested that cartilage tissue is generated with cultured chondrocytes up to P2 but not beyond P4. Northern blot analysis is useful for the assessment of whether the cells are capable of regeneration.

Absorbable Implants↗

Biodegradable interlocking nails for fracture fixation.

Serious problems such as stress shielding, allergic reactions, and corrosion are associated with the use of metallic fracture fixation devices in fractured long bones. Metal implants often are removed during a second retrieval operation after fracture healing has completed. A biocompatible implant that degrades slowly during implantation would obviate the need for a second operation and save the patient from considerable physical, psychologic, and financial discomfort. The biodegradable implant must provide the fractured limb sufficient support for a certain time, allowing early loading. A gradual transfer of load from the biodegradable implant to the bone would result in a better product of bone healing and avoid stress shielding. In an animal model using adult sheep, two types of biodegradable polymer interlocking nails were tested in comparison with a stainless steel interlocking nail. Fracture healing, mechanical properties of the bones, degradation behavior in vivo and in vitro, and tissue response were monitored during a 2 1/2-year followup study. To detect shifts in acid base relations caused by the release of acid compounds, pH measurements were performed. Fracture healing was unimpaired, and the mechanical test results of all three groups were excellent. Histologic analysis showed a mild inflammatory response, but no pH shifts were observed. The results of this study justify additional research on these promising materials.

Absorbable Implants↗

Characteristics of tissue-engineered cartilage on macroporous biodegradable PLGA scaffold.

BACKGROUND: The purpose of this study was to establish in vivo culture of chondrocytes on biodegradable, poly-D,L-lactic-co-glycolic acid (PLGA) scaffolds and to analyze the characteristics of the reconstructed cartilage. METHODS: In vitro cultured chondrocytes that were grown on a polyhydroxyethyl methacrylate (poly-HEMA) coated dish were seeded onto the PLGA scaffolds to make a cell-polymer construct before implantation. One cell scaffold construct was carefully implanted in the subcutaneous pocket of a nude mouse and another cell-free scaffold was implanted in the opposite side of the same nude mouse as the control. Morphologic, biochemical, and immunohistochemical characteristics of cells cultured within the PLGA constructs were examined after 8 weeks and 16 weeks of harvesting in the nude mouse. RESULTS: New cartilage began to be generated in the period of 8 weeks and the neocartilage formation was accomplished in 4 months with the exact dimensions of the original scaffold in this in vivo study. All the explants showed the irregular shape of viable chondrocytes within normal lacunae and a mature cartilaginous matrix, and they positively immunostained for collagen type II. CONCLUSION: The new tissue-engineered cartilage in vivo on PLGA scaffolds displayed the biochemical characteristics of cartilage tissue, and it showed chondrocyte-specific phenotypes and morphology that were similar to the native cartilage.

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Three-dimensional cytoarchitecture of angiogenic blood vessels in a gelatin sheet implanted in the rat skeletal muscular layers.

To demonstrate the structure of angiogenic blood vessels three-dimensionally, a gelatin sponge sheet immersed in a vascular endothelial growth factor (VEGF) solution was implanted in the rat dorsal muscular layer, and examined by light microscopy and scanning electron microscopy (SEM) 5 days to 2 weeks after implantation. Light microscopy of anti-collagen IV antibody immunostained specimens enabled a determination of the basement membrane tube of newly formed blood vessels in the implanted sponge sheet. The tubes were 5-40 microm in diameter, and sometimes tapered to a slender cord within the vascular network. The SEM study of 30% KOH treated tissues revealed two types of tapering ends of newly formed blood vessels. One consisted of endothelial cells with microprojections, and lacked any investment of pericytes over the length of 5-20 microm. The other type was a tapering tip of the endothelial tube covered with pericytic processes. The presence of long processes of pericytes extending beyond the tip of the endothelial tube and connecting to the adjacent vessel wall indicates that this type was produced by endothelial tube regression. Thus, the present study supports the ideas that endothelial tube formation is followed by pericyte coverage at the sprouting tip, and that endothelial tube regression precedes pericyte detachment at the regressing site.

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