The influence of human amniotic fluid on the potential of rabbit ear perichondrial flaps to form cartilage tissue.

Since several experimental and clinical studies demonstrated the chondrogenic potential of perichondrium, there has been great interest in examining factors that might promote neochondrogenesis from perichondrium. Human amniotic fluid contains hyaluronic acid, growth factors and extracellular macromolecules, and may, therefore, have a stimulating effect on cartilage regeneration. This experimental study investigated the effect of human amniotic fluid on cartilage regeneration from rabbit ear perichondrial flaps, using 96 ears of 48 New Zealand young rabbits. A perichondrial flap was elevated and a cartilage defect measuring 20 mm x 15 mm was created on the dorsum of each ear, then the perichondrial flap was sutured in place. The ears were divided into two groups according to the solution injected underneath the perichondrial flap. The right ears, which were injected with 0.2 ml human amniotic fluid, formed the experimental group, and the left ears, which were injected with 0.2 ml saline, formed the control group. Macroscopic and histological progression of neochondrogenesis were evaluated at 2, 4, 6 and 8 weeks after surgery. Macroscopically, the cartilage in the experimental group was generated quickly and had a similar appearance to the surrounding cartilage tissue, whereas in the control group minimal cartilage formation was observed at 4 weeks. Histologically, the neocartilage was significantly thicker in the experimental group than in the control group at 8 weeks (P < 0.05, Student's t -test). It can be concluded that human amniotic fluid enhances new cartilage formation from rabbit ear perichondrial flaps. The preventive effect of human amniotic fluid on scar formation and the rich content of growth factors and extracellular matrix precursors may play a role in this result.     

The development of bone after perichondrial grafting: an experimental study using ear and rib perichondrium in rabbits

The development of bone after perichondrial grafting was investigated using rabbit ear and rib perichondrium. Sixty-four white adult female rabbits were used. Both free and vascularised perichondrial grafts were undertaken. In each case the chondrogenic potential of perichondrium was proved. Furthermore, when the perichondrium was vascularised or grafted in recipient sites having good blood circulation, the development of large areas of bone was observed around the regenerated cartilage.     

Inhibitory effect of mature cartilage on perichondrial neochondrogenesis

The perichondrium adhering to mature cartilage is not active, but that separated from cartilage is highly chondrogenic. Cartilage formation from isolated perichondrium does not last forever, and perichondrium soon becomes inactive. What activates or inactivates the perichondrium. The authors investigated the effect of mature cartilage on the cartilage formation from perichondrial graft material. The results showed that mature cartilage attached to the perichondrium inhibited neochondrogenesis. The phenomenon that cartilage--a product of chondrogenesis--inhibits neochondrogenesis of perichondrium can be called negative feedback.     

Repair of cartilage defects with periosteal grafts.

Alternative sources for repair of cartilage defects are limited and donor sites are associated with morbidity. It is known that cartilage development from periosteal grafts is possible. Various factors have been found positively to affect this process in experimental settings. However, all of these studies were limited to joint cartilage. We conducted an experimental study in rabbits for the investigation of the elastic cartilage regeneration from perichondrial and periosteal grafts together with the effects of hyaluronan on this process. 1 x 1 cm(2) cartilage defects were created on the elastic ear cartilage of rabbits. Four experimental groups with 18 ears in each group were created: Group 1 (repair with perichondrium graft), group 2 (repair with periosteum graft), group 3 (repair with periosteum graft+hyaluronan), group 4 (defect-only control group). Macroscopic and microscopic evaluations were done on the 4th, 8th and 12th weeks. Cellular morphology of the regenerated cartilage and its integration and similarity with adjacent cartilage were evaluated. Cartilage regeneration groups 1, 2 and 3 were found to be statistically different from the control group. There was not a significant difference between groups 1 and 2 or 2 and 3. There is no significant difference between perichondrial and periosteal grafts in cartilage regeneration, and hyaluronan has no beneficial effect on this process.     

Effect of lyophilized heterologous collagen on new cartilage formation from perichondrial flaps in rabbits: an experimental study

New cartilage formation originated from perichondrium has previously been researched in many clinical and experimental studies. 1-6 After these studies, the use of perichondrium for the repair of cartilage defects has been used clinically when enhancing neochondrogenesis has been found to be of great value in decreasing the recovery period. Collagen is the major protein of whole connective tissue, and in vitro positive effects of collagen matrices on neochondrogenesis have also been studied before. 7-9 In this experimental study, in vivo effects of heterologous collagen sponge in perichondrial neochondrogenesis were examined in an animal model, and acceleration and enhancement effects were observed.     

The effect of growth factors and synovial fluid on chondrogenesis in perichondrium

Reconstruction of cartilage with perichondrium depends on the chondrogenic property of the perichondrial fibrocytes. The present investigation concerns the conditions for the differentiation of fibrocytes into chondrocytes both in vivo and in vitro. For the in vivo studies specimens of rib and auricular perichondrium from adult rabbits were wrapped round silicon rods which were enclosed in dialysis bags. One was placed in the suprapatellar pouch of the knee joint and one was placed intraperitoneally in each rabbit. After two months the bags were extracted, the perichondrium prepared for microscopic examination, and the chondrogenesis evaluated. In vitro the perichondrium was divided into small pieces and incubated with tissue culture medium. The medium was supplemented with fetal calf serum, together with epidermal growth factor, platelet derived growth factor, synovial fluid, or with human serum albumin (control group). After three weeks the explants were prepared for microscopy. Chondrogenesis was judged by the degree of cellular enlargement, capsule formation, deposition of matrix, and activation of the outer fibrocytic layer. In vivo, good cartilage development was found in all specimens placed in the knee joint but, in those placed intraperitoneally, little if any chondrogenesis was seen. In vitro profound differentiation occurred in all cultures supplemented with epidermal growth factor and platelet derived growth factor. An equivalent differentiation was found in perichondrium that had been incubated with synovial fluid. We conclude that the differentiation of perichondrial fibrocytes is initiated in vitro by growth factors. In addition, we have shown that synovial fluid contains factors that promote and enhance the development of cartilage from perichondrium.     

An experimental model for ear reconstruction with moulded perichondrial flaps: a preliminary report

Attempts to create a pinna by moulding cartilage fragments have been reported previously by Peer. The regenerative capabilities of perichondrium are well known. Combining these concepts, we succeeded in creating a cartilage pinna by implanting perichondrial flaps between the leaves of a methylmethacrylate mould. Ten rabbits were divided into two groups, the first using vascularised perichondrial flaps and the other using free perichondrium into which an arterial and venous pedicle was implanted. Both of these preparations were placed in the moulds in a subcutaneous pocket. Six weeks later, these constructions were harvested and histological examination conducted. Active cartilage growth was noted in both groups. The construction was completed by coverage with a split thickness skin graft. Clinically, this new procedure may provide a means of pinna reconstruction with a thin flexible cartilage framework, with excellent relief.     

Cartilage formation from perichondrium in a diffusion chamber

Autologous perichondrial grafts from rabbits' ears in diffusion chambers resulted in the formation of cartilage. This clearly revealed that neither blood clots nor the ingrowth of capillaries is essential for perichondrial neochondrogenesis.     

Neochondrogenesis in free intraarticular periosteal autografts in an immobilized and paralyzed limb. An experimental investigation in the rabbit

The purpose of this investigation was to determine whether neochondrogenesis can be induced in free intraarticular autografts of periosteum in the complete absence of motion. In 17 adolescent rabbits, a rectangular graft of periosteum was elevated from the medial aspect of each proximal tibia and folded back on itself so that its deep (cambium) layer was facing outward on both sides. The grafts were transplanted into both ipsilateral knee joints that had been paralyzed by section of the femoral and sciatic nerves, and a cast was applied to one hind limb to provide immobilization. The opposite knee joint of each animal was then placed in the continuous passive motion (CPM) apparatus. At the time of death (21 days postoperatively), some degree of neochondrogenesis was evident in 69% of the grafts in the group with casts and in 100% of the grafts in the CPM group. Hyaline cartilage was the predominant tissue in 13% of the grafts in the group with casts compared to 63% of the grafts in the CPM group. Although this investigation has confirmed the chondrogenic potential of free periosteal grafts in a synovial fluid environment (and the significantly stimulating effect of CPM), the results have also demonstrated that at least some hyaline cartilage can be formed by periosteal grafts even with paralysis of the limb plus immobilization of the joint (presumably complete immobilization). Thus, other factors capable of stimulating neochondrogenesis warrant investigation.     

Rib perichondrial grafts for the repair of full-thickness articular-cartilage defects. A morphological and biochemical study in rabbits

The purpose of this study was to investigate the use of perichondrial grafts in articular cartilage defects and to characterize the newly formed cartilage. In a rabbit model, rib perichondrium was used to repair full-thickness defects in the femoral condyle. The quality of repair was then evaluated histologically and biochemically at six and twelve weeks after grafting. Unacceptable results were obtained in 50 per cent of the rabbits. These failures were due to condylar fracture in 20 per cent, failure of graft attachment in 20 per cent, and infection in 10 per cent. The technique of grafting must be improved to increase the percentage of successful grafts in which neocartilage with a relatively normal chemical composition fills the articular cartilage defect. Successful grafts proliferate to fill the full-thickness defect with neocartilage, which has biochemical characteristics that are similar to those of hyaline cartilage.     

Comparative Study of the Reconstruction of Articular Cartilage Defects with Free Costal Perichondrial Grafts and Free Tibial Periosteal Grafts: An Experimental Study on Rabbits

The purpose of this study was to compare the chondrogenic potential of free perichondrial with free periosteal grafts in the resurfacing of full-thickness defects of patellar articular cartilage in rabbits. We used adolescent New Zealand rabbits weighing between 2.4 and 3.6 kg. A 6-mm wide and 3-mm thick defect was created on the patellar articular surface. A total of 30 rabbits were randomly divided into a control group and two test groups. One test group received free perichondrial grafts (PC); the other received free periosteal grafts (PO). All the animals were killed 8 weeks after surgery. All the histological samples were scored from 0 to 17 according to a standard scoring system. Differences in the quality of the regenerated tissue were only found between the control and the test groups. There were no statistically significant histological differences between the grafted defects of the PC and the PO groups that there are not on any of the variables. The results of this study support that there are not significant differences in the quality of the repair tissue when using these two types of biological grafts. Received: 7 July 1998 / Accepted: 8 January 1999     

Shaped regeneration of rabbit ear perichondrium.

The neocartilage that regenerated from the ear perichondrium in 18 growing rabbits was shaped using biodegradable implants. The animals were divided into two groups according to the operation used. In group 1 a self-reinforced polyglycolic acid (SR-PGA) rod was placed inside a perichondrial pocket on the dorsum of the ear and surrounded by blood clot, and in group 2 a flap of ear perichondrium was shaped around an SR-PGA rod. Samples were taken for histological examination six weeks after the operation. Active growth of neocartilage was seen in both groups and the SR-PGA rod had successfully guided the perichondrial regeneration in most of the animals.     

Reconstruction of Articular Cartilage with Free Autologous Perichondrial Grafts

An experimental study in adult rabbits has been performed to find out whether the cartilage forming capacity of the perichondrium could be utilized in reconstruction of articular cartilage. The normal articular cartilage of the glenoid surface of the humero-scapular joint was completely removed. Auricular perichondrium was grafted to cover the exposed bony surface with the active chondrogenic layer of the perichondrial graft facing the joint cavity. The joint was not immobilized but the operated limb was amputated at wrist level to avoid weight bearing. The animals were sacrificed at different time intervals ranging from 1 to 17 weeks. In 12 out of 14 grafted rabbits regeneration of cartilage occurred. In 6 of 10 control cases where no perichondrium was grafted to cover the resected surface no cartilage was found. In the other 4, only small areas of mature cartilage were seen, probably remnants of the original articular cartilage.     

Degree of differentiation of chondrocytes and their pretreatment with platelet-derived-growth factor. Regulating induction of cartilage formation in resorbable tissue carriers in vivo

Current methods for articular cartilage repair are unpredictable with respect to clinical success. In the present study, we investigated the ability of cells from articular cartilage, perichondrium, and costochondral resting zone to form new cartilage when loaded onto biodegradable scaffolds and implanted into calf muscle pouches of nu/nu mice. Prior in vitro studies showed that platelet derived growth factor-BB (PDGF-BB), but not transforming growth factor beta-1 (TGF-beta 1), basic fibroblast growth factor, or bone morphogenetic protein-2 promoted proliferation and extracellular matrix sulfation of resting zone chondrocytes without causing the cells to exhibit a hypertrophic chondrocyte phenotype. TGF-beta 1 has also been shown to stimulate chondrogenesis by multipotent chondroprogenitor cells like those in the perichondrium. In addition, PDGF-BB has been shown to modulate chondrogensis by resting zone cells implanted in poly(D,L-lactide-co-glycolide) (PLG) scaffolds. In the present study we examined whether the cartilage formation is dependent on state of chondrocyte maturation and whether the pretreatment of chondrocytes with growth factors has an influence on the cartilage formation. Scaffolds were manufactured from 80% PLG with a 75:25 lactide:glycolide ratio and 20% modified PLG with a 50:50 lactide:glycolide ratio (PLG-H scaffolds). For each experimental group, four nude mice received two identical implants, one in each calf muscle resulting in an N = 8 implants: PLG-H scaffolds alone; PLG-H scaffolds with cells derived from either the femoral articular cartilage, costochondral periochondrium, or costochondral resting zone cartilage of 125 g male Sprague-Dawley rats; PLG-H scaffolds with either articular chondrocytes or resting zone chondrocytes that were pretreated with 37.5 ng/ml rhPDGF-BB for 4 h or 24 h before implantation, or with perichondrial cells treated with PDGF-BB plus 0.22 ng/ml rhTGF beta-1 for 4 h and 24 h. At 4 or 8 weeks after implantation, samples were harvested and analyzed histomorphometrically for new cartilage formed, area of residual implant and area of fibrous connective tissue. Only resting zone cells showed the ability to form new cartilage at a heterotopic site in this study. There was no neocartilage found in nude mice with implants loaded with either articular chondrocytes or perichondrial cells. Pretreatment of resting zone chondrocytes for 4 h prior to implantation significantly increased the amount of newly formed cartilage after 8 weeks and suppressed chondrocyte hypertrophy. The amount of fibrous connective tissue around implants containing either articular chondrocytes or perichondrial cells decreased with time, whereas the amount of fibrous connective tissue around implants containing resting zone chondrocytes pretreated with PDGF-BB was increased. The results showed that resting zone cells can be successfully incorporated into biodegradable porous PLG scaffolds and can induce new cartilage formation in a nonweight-bearing site. Articular chondrocytes as well as perichondrial cells did not have the capacity for neochondrogenesis when implanted heterotopically in this model.     

Reconstruction of auricular framework using autologous perichondrial grafts. An experimental study]

An experimental study was performed in 13 adult dogs to find out whether the cartilage forming capacity of the costal perichondrium could be utilized in reconstruction of the auricular framework. Silastic ear-shaped frameworks, which were wrapped by free costal perichondrial grafts, were transferred to the subcutaneous space on both side of the thorax. The animals were sacrificed at 2 months, 3 months, and 4 months postoperatively. After 6 weeks, the reconstructed auricular cartilage framework collapsed and the silicone framework was rejected. In groups of 2, 3 and 4 months, collapse of the cartilage framework occurred when the silicone tube was removed in 7 day. There was certain elasticity, but the auricular framework had only little resistance to pressure. How to improve new cartilage production would be the key point of further study. Histologically, it was shown that mature hyalin cartilage was generated in 2 months postoperatively.     

The potential of adult human perichondrium to form hyalin cartilage in vitro

The usefulness of adult human perichondrium for the restoration of articular cartilage defects depends on the potential to form hyalin cartilage. In order to evaluate the capacity of adult human perichondrium to form hyalin cartilage in vitro, perichondrium of the rib of eight adult human beings was cultured in vitro. After removal of residual cartilage, perichondrial explants were cultured for 7 or 10 days. The explants were histologically examined using specific stains to prove the presence of glycosaminoglycans (GAGs) normal for hyalin cartilage. Clear differentiation of perichondrial cells towards chondrocytes was noted. The chondrocytes synthesized new matrix substances normally present in hyalin cartilage. This investigation supports the usefulness of adult human rib perichondrium for the restoration of cartilage defects. Due to the enormous potential of the rib perichondrium to form hyalin cartilage in vitro, even defects in joints with a rather thick cartilage layer might be restored using this biological material.     

Effect of human amniotic fluid on bone healing

BACKGROUND: Bone healing continues to pose challenges for researchers and clinicians working in the field of plastic surgery. Complete bone regeneration cannot be obtained in critical size osseous defects without the application of osteogenic or osteoinductive bone material. In this study, we hypothesized that because extracellular matrix components are known to play a major role in the first steps of healing during bone or injury healing and because hyaluronic acid as chondroitin sulfate is recognized as an osteogenic compound without osteoinductive activity, human amniotic fluid, which contains high concentrations of hyaluronic acid, gyaluronic acid -stimulating activator, and other factors, might accelerate bone healing when applied subperiosteally to rabbit calvarial defects. MATERIALS AND METHODS: We created 20 calvarial defects in 10 12-week-old New Zealand white rabbits who were divided into 2 groups. Group 1 defects were instilled with human amniotic fluid, whereas the group with contralateral defects, i.e., group 2, were given with same amount of normal saline solution. We then measured the density of the bone that formed over the defects using computed tomography at the third, fourth, fifth, and sixth weeks postoperatively. After this period, the defects were harvested for histopathologic evaluation. RESULTS: The defects from group 1, which were treated with human amniotic fluid, showed significantly higher ossification than the group 2 defects, which were instilled with saline solution. Histological examination at 6 weeks postoperatively revealed that the defects treated with human amniotic fluid (group 1) had superior ossification compared with the control group defects (group 2). CONCLUSION: Because of its positive effects on bone healing and also because of its ability to be stored in deep freeze if made cell-free, human amniotic fluid would appear to be a useful adjunct in the treatment of bone healing.     

Cartilage grafts—present status

Cartilage grafts have been in use for almost a century and have proved their usefulness. Many questions about immunology, survival, growth, and role of perichondrium are still debated. We are presenting a review of the literature and of our experimental work on cartilage grafts. Ear cartilage was transplanted from 28 young New Zealand rabbits subcutaneously in the chest walls. The grafts were divided into 1) bare cartilage, 2) perichondrium, 3) cartilage covered with perichondrium on one side, and 4) cartilage covered with perichondrium on both sides. The grafts were measured in length and weight before transplantation, and at 2 and 4 months after transplantation; they were compared to a control piece of cartilage tagged in situ. Histologic examination was performed on all retrieved grafts. Transplanted cartilage survival was good in over 75% of cases; however, cartilage production from perichondrium was minimal and no growth was noticed in any of the grafts.     

Cartilage grafts--present status

Cartilage grafts have been in use for almost a century and have proved their usefulness. Many questions about immunology, survival, growth, and role of perichondrium are still debated. We are presenting a review of the literature and of our experimental work on cartilage grafts. Ear cartilage was transplanted from 28 young New Zealand rabbits subcutaneously in the chest walls. The grafts were divided into 1) bare cartilage, 2) perichondrium, 3) cartilage covered with perichondrium on one side, and 4) cartilage covered with perichondrium on both sides. The grafts were measured in length and weight before transplantation, and at 2 and 4 months after transplantation; they were compared to a control piece of cartilage tagged in situ. Histologic examination was performed on all retrieved grafts. Transplanted cartilage survival was good in over 75% of cases; however, cartilage production from perichondrium was minimal and no growth was noticed in any of the grafts.     

Growth of two types of cartilage after implantation of free autogeneic perichondrial grafts

The perichondrium of adult rats was dissected from the posterior side of the ear where a plane of separation can be easily found between the superficial chondrocytes and the rest of the cartilage. When pulled off, the perichondrium brings with it a cartilaginous strip adhered to its inner layer, with the detachment surface showing projections of broken capsular matrix (PBCM). The perichondrium and subperichondrial cartilage were then transferred as autogeneic grafts to preformed muscle pockets of the abdominal wall and to everted vein chambers placed free in the iliac blood flow. During a period of one to 12 days, chondrogenesis was studied in the grafts and in the graft bed areas next to subperichondrial cartilage. When the perichondrium was placed into a muscular pouch, wherein perichondrocytes survived and a prominent vascular ingrowth in the graft bed was observed, the presence of two types of newly formed cartilage was demonstrated (Types I and II). These types showed differences in their location, time of appearance, and microscopic characteristics. Type I neocartilage appeared in the inner layer of the perichondrium on the third or fourth day after grafting; at this time the cells, surrounded by a well-defined capsular matrix, were large, darkly stained, and highly electron dense. Type II neocartilage, separated from Type I by the PBCM, appeared in the graft bed area located within perichondrial folds on the sixth or seventh day after implantation. Their cells showed a poorly defined capsular matrix and were smaller, lighter stained, and less electron dense than those of Type I. When the perichondrium was transplanted to everted vein chambers placed in the iliac blood flow, wherein perichondrocytes survived and vascular ingrowth from the graft bed was not present, Type I neocartilage was formed but Type II was not. The morphologic and histoautoradiographic findings in these studies suggest that Type I cells come from perichondrocytes of the inner perichondrial layer, whereas Type II cells originate from the undifferentiated perivascular mesenchymal cells of the graft bed.