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.     

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.     

Rib perichondrial autografts in full-thickness articular cartilage defects in rabbits.

This study investigated the resurfacing of full-thickness articular defects in the adult rabbit medial femoral condyle using a rib perichondrial graft. The graft was secured to a 4-mm-diameter bone core removed from the femoral condyle. Two postoperative treatment protocols were studied: one group had ad libitum cage activity (CAGE; n = 95) and the other group received two weeks of passive motion (PM; n = 73; eight hours per day, five days per week) followed by cage activity. Animals in both groups were killed at six, 12, 26, and 52 weeks. Repair tissue resembling hyaline cartilage formed in a majority of animals at all time periods and in both postoperative treatment groups. The overall success rates in which repair tissue formed were 58% in the CAGE group and 56% in the PM group. However, over time, a maturation of the neocartilage into nearly normal hyaline articular cartilage was noted with the percentage of Type II collagen increasing from 55% at six weeks to 82% at one year. The complex shear modulus of the repair tissue for both groups became similar to normal cartilage with increased healing time. There was no statistical difference in shear moduli between the two treatment modalities. These results show that cartilage repair tissue derived from rib perichondrium could mature into hyaline articular cartilage over time and would not degrade by one year after repair.     

Effects of human amniotic fluid on peripheral nerve scarring and regeneration in rats

OBJECT: Peripheral nerve repair surgery is still replete with challenges. Despite technical improvements in microsurgery, classic methods of nerve repair have failed to provide satisfactory results. The purpose of this study was to investigate the effects of amniotic fluid from humans on peripheral nerve scarring and regeneration in rats. METHODS: Forty adult Sprague-Dawley rats were used in this study. After the right sciatic nerve in each rat was transected and repaired using an epineural suture procedure, the nerves were divided into two groups according to the solution applied around the repair site: experimental group, 0.3 ml human amniotic fluid (HAF); and control group, 0.3 ml saline. Macroscopic and histological evaluations of peripheral nerve scarring were performed 4 weeks postsurgery. Nerves treated with HAF demonstrated a significant reduction in the amount of scar tissue surrounding the repair site (p < 0.05). No evidence of a reaction against HAF was noted. Functional nerve regeneration was measured once every 2 weeks by using a sciatic function index until 12 weeks postsurgery. Functional recovery in nerves treated with amniotic fluid occurred significantly faster than that in nerves treated with saline (p < 0.05). Peripheral nerve regeneration was evaluated histomorphologically at 12 weeks postsurgery. Nerves treated with amniotic fluid showed significant improvement with respect to the indices of fiber maturation (p < 0.05). CONCLUSIONS: Preliminary data show that HAF enhances peripheral nerve regeneration. The preventive effect of HAF on epineural scarring and the rich content of neurotrophic and neurite-promoting factors possibly contribute to this result.     

Formation of chondroitin sulphate proteoglycan in cartilage regenerated from free perichondrial graft

Perichondrium from rabbit auricular or rib cartilage was used as a free autogenous graft and transplanted either to the subcutaneous tissue of the back of the rabbit or to an experimental defect in the femur condyles. Outgrowth of new tissue, morphologically indistinguishable from cartilage, was observed after six weeks. Inorganic 35SO4, administered in vivo, was incorporated into the newly formed tissue. The labelled products were isolated, identified, and compared with those obtained from authentic cartilage of auricular, rib or joint surfaces. The products of newly formed cartilage were similar to those of authentic cartilage. The results support earlier morphological findings, indicating that perichondrium from rib cartilage has a better ability to regenerate than auricular perichondrium. The synovial environment seems to have a positive effect on the generation of cartilage.     

Long-term results of rib perichondrial grafts for repair of cartilage defects in the human knee

Summary. Eighty-eight patients with articular cartilage defects in the knee were treated by perichondrial arthroplasty between 1986 and 1992. An autogenous strip of costal perichondrium was fixed in place with fibrin glue, followed by immobilisation, continuous passive motion, and partial weightbearing. The results were evaluated using the Hospital for Special Surgery Score for knee function, radiographs, arthroscopy and the patient’s subjective opinion. The results after a mean follow-up of 52 months were good in 38%, fair in 8% and poor in 55%. Previous drilling or shaving of a defect, concomitant osteoarthritis, older age and a long history of complaints proved to be contraindications. Good results were seen in 91% of isolated defects. Perichondrial arthroplasty can be beneficial in the repair of cartilage defects. It will reduce symptoms in carefully selected cases, and avoid more extensive operations for osteoarthritis.

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Résumé. Depuis septembre 1986 jusqu’à decembre 1992 quatre-vingt huit patients presentant les lésions cartilagineuses au genou ont été traité par arthroplastie périchondrale. Dans les lésions, une partie du périchondre de la c?te a été fixé avec de la colle de fibrine. Les soins consécutifs consistaient à une immobilisation, une mobilisation passive, et a une mise en charge partielle puis complète. L’évaluation des résultats a été fait avec l’aide de la cotation Hospital for Special Surgery pour l’articulations du genou, par examen radiologique, arthroscopie postoperatoire et recueil de l’opinion subjective du patient. Les résultats après une étude moyenne de cinquante deux mois ont été bon à 38%, modéréà 8% et mauvais à 55%. Les contre-indications pour cette technique sont des forages ou nettoyages précédents, l’arthrose, l’age avancé du patient, et des douleurs préexistantes. Des bons résultats sont vus dans les lésions isolées à 91% (30/33). Nous concluons que l’arthroplastie avec périchondre peut être favorable pour réparer des lésions cartilagineuses. Cette technique peut diminuer les douleurs des patients bien sélectionnés et prévenir les opérations plus compliquées pour arthrose.

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Accepted: 13 December 1996     

Pedunculated synovium grafts in articular cartilage defects in rabbits.

A rabbit model was used to assess the nature of healing tissues in hyaline cartilage defects and to compare the healing in defects treated with pedunculated synovium grafts to those in defects without synovial grafting. Both knees of 28 1-year-old rabbits were operated. A 3 x 2-mm cartilage defect that exposed cancellous bone was created in the non-weight-bearing area of each medial femoral condyle. Each right-knee defect was covered with a pedunculated synovial graft obtained from the same joint, and the left-knee defects were left uncovered as controls. Groups of rabbits were sacrificed at 3, 6, 12, and 24 weeks postsurgery. Sections from each knee were stained with hematoxylin-eosin and safranin O-fast green staining, and were immunohistochemically stained for type II collagen. The healing at each site was histologically scored, and the intensity of staining for type II collagen was graded. At 12 and 24 weeks, statistical comparisons of histological scores revealed significantly more hyaline cartilage tissue in the synovium-grafted defects. At 24 weeks, these same defects showed significantly more type II collagen. Thus, pedunculated synovium transplantation appears to hold promise as a method for repairing hyaline cartilage defects.     

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.     

Histological and biochemical evaluation of perichondrial transplants in human articular cartilage defects.

From 1986 to 1992, 88 patients with articular defects in the knee were treated with a perichondrial arthroplasty. In this study, we report on the results for 22 biopsies of grafted tissue with a mean follow-up of 21 months. Biopsies were obtained at routine arthroscopy after approximately 1 year or at arthroscopy or arthrotomy at a later stage when patients were operated on again because of recurrent complaints. Biopsies were taken only when a partial failure was present or when there was a clear failure resulting in fibrocartilage, a loose flap, or a loose body. The biopsies were analyzed histologically, biochemically for the amount of type-II collagen, and immunohistochemically with antibodies for types I, II, and X collagen. The well-being of the patients was investigated with use of the Hospital for Special Surgery knee score. The biopsies from 6 patients contained more than 50% hyaline cartilage. At arthroscopy, the mean relative amount of type-II collagen was 56% in the biopsies classified as good. The cartilage of the grafted area was macroscopically normal for eight of the 22 biopsies. Histological and biochemical analysis of biopsies from failed transplants showed fibrocartilage with mainly type-I collagen. These tissues were retrieved primarily from patients with additional abnormalities in the knee joint. It was concluded that adult human perichondrium is able to form hyaline-like cartilage in an isolated cartilage defect in an otherwise healthy knee.     

Effects of human amniotic fluid on peritendinous adhesion formation and tendon healing after flexor tendon surgery in rabbits

The effect of the topical application of human amniotic fluid (HAF) on peritendinous adhesion formation and tendon healing was investigated in 32 New Zealand adult rabbits. The long flexor tendons of the digits of each hind paw were completely divided and repaired with a modified Kessler technique. The rabbits were randomly divided into 4 experimental groups according to the type of repair used: sheath excision, sheath excision and local HAF application, sheath repair, and sheath repair and local HAF application. The extent of adhesions and the healing status of the tendons were macroscopically and histologically evaluated at 12 weeks. Tensile strength of the repaired tendons was measured biomechanically at 20 weeks. The least adhesion and the best healing were observed in tendons treated with sheath repair and HAF application. Tendons treated with HAF had significantly higher tensile load values. Topical application of HAF immediately after tenorrhaphy is significantly effective in preventing peritendinous adhesion formation without impairment of tendon healing in this rabbit model.     

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.     

The role of polyglycolic acid rods in the regeneration of cartilage from perichondrium in rabbits.

The role of polyglycolic acid (PGA) rods in the regeneration of cartilage from perichondrium was investigated in 12 growing rabbits. The fifth rib cartilage was resected subperichondrially from both sides. A 10 X 1.5 mm self-reinforced polyglycolic acid (SR-PGA) rod was inserted on one side to replace the resected cartilage and the retained perichondrium was sutured around the implant. On the control side the perichondrium was shaped into a tube without an implant. Samples were taken 4, 12, and 20 weeks after operation. Pronounced neocartilage formation was seen on both sides, and had grown to form a tube around the implant. Also new bone formation was seen in 12 and 20 weeks. Foreign body reaction was seen inside the implants in every animal.     

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 articular defects by perichondrial grafts. Experiments in the rabbit

A defect was created in the articular cartilage of the rabbit knee leaving the subchondral bone intact. The lesion was repaired by an autologous graft of costal perichondrium and fixed with fibrin glue. The result was compared with a nontreated defect in the contralateral knee. In 26 out of 30 knees, graft fixation proved to be adequate. In the grafted group the perichondrium developed macroscopically and histologically into normal hyaline cartilage. The nongrafted defects showed only limited repair.     

人羊水来源干细胞参与小鼠损伤肌肉修复的实验研究

目的研究人羊水来源干细胞(human amniotic fluid colony derived stem cells,hAFCSCs)是否参与小鼠肌肉损伤修复过程,探讨应用hAFCSCs治疗肌肉损伤的方法及可行性。方法 B超引导下穿刺抽取人孕中期羊水,体外分离、培养得到hAFCSCs,取第6～8代细胞备用,同时提取总RNA行RT-PCR鉴定干细胞相关基因。取16只6～8周龄Nod/Scid小鼠,体重20～24 g,利用心肌毒素联合X线照射建立双侧胫骨前肌肌肉损伤模型,右侧胫骨前肌内注射hAFCSCs(3.3×107个/mL)30μL作为实验组,左侧胫骨前肌同法注射等量完全培养液(含15%FBS、18%Chang B、2%Chang C、1%青链霉素、1%L-谷氨酰胺的α-MEM培养基)作为对照组。细胞移植术后2、4周各处死小鼠8只,取材行肝细胞生长因子受体(c-Met)、成肌调节因子(Myf-5)、层粘连蛋白(Laminin)、结蛋白(Desmin)与人特异性细胞核有丝分裂器(NuMa)组织免疫双重荧光染色观察。结果原代hAFCSCs培养5～7 d后可见细胞克隆形成;8～10 d后可挑取细胞形态均一的克隆进行稳定传代,6～8代后可获得足量的干细胞。RT-PCR检测示所获得hAFCSCs体外扩增培养至第6代仍表达干细胞相关基因。细胞移植术后2周免疫双重荧光染色示,实验组胫骨前肌内检测到NuMa表达,未检测到hAFCSCs表达肌源性细胞相关表型;术后4周,实验组胫骨前肌内检测到NuMa表达,部分细胞同时表达NuMa和c-Met、Myf-5,部分肌纤维同时表达NuMa和Desmin、Laminin。术后2、4周,对照组胫骨前肌均未检测到NuMa表达。结论 hAFCSCs体外培养后移植到小鼠肌肉损伤部位,可参与损伤肌肉的修复过程。     

Histological assessment of skin grafts stored in amniotic fluid and saline

We investigated the efficacy of amniotic fluid as a substance in which to store grafts; it is rich in nutrients, proteins, and growth factors, and has well-known antimicrobial features. We compared it with the widely-used and practical saline. Split-thickness grafts 4 × 4 cm were prepared from the back of 20 rats and divided into four groups (n = 5 each). The rolled grafts were wrapped in gauze dampened with saline or amniotic fluid and placed into refrigerators in sterile containers for storage. On days 7, 14, 21, and 28, histological examinations were made. A semiquantitative evaluation of the histological damage to the skin was made by scoring its degree of severity. Compared with saline, histological scores in the grafts stored in amniotic fluid were found to be significantly lower on the 14th, 21st, and 28th days (p values on days 14, 21, and 28; cell swelling: 0.014, 0.006, and 0.005, respectively; nuclear swelling: 0.003, 0.006, and 0.007, respectively; nuclear pleomorphism: 0.004, 0.005, and 0.003, respectively; nuclear haloes: 0.015, 0.005, and 0.005, respectively; nuclear pyknosis: 0.003, 0.005, and 0.003, respectively; dermo-epidermal clefting: 0.005, 0.003, and 0.003, respectively; eosinophilia and mitosis: 0.003, 0.006, and 0.004, respectively; dermal collagen: 0.003, 0.003, and 0.003, respectively). Amniotic fluid maintained preservation better for skin grafts than saline. Comparison with other modern storage media would be beneficial.