The importance of procedure specific training in harvesting periosteum for chondrogenesis

This study was performed to determine the influence of procedure specific and nonspecific training on the chondrogenic potential of explanted periosteum. Seven operators, with varying degrees of orthopaedic surgical experience and procedure specific training in periosteal harvesting, harvested 10 to 16 periosteal explants each from the proximal medial tibiae of 42 New Zealand White rabbits that were 2 months of age. The chondrogenic index assay involved culturing the explants in agarose suspension for 6 weeks, followed by computerized histomorphometric analysis. Chondrogenic indices (the average percent area of cartilage grown in the cultured explants) ranged from 12% to 81% and were influenced strongly by each operator's experience with the technique of periosteal harvesting. Average cartilage yields before practice were in the range of 12% +/- 4% for a technician and 44% +/- 6% for a surgeon, compared with 54% +/- 7% and 79% +/- 2%, respectively, after practice involving more than 300 explants each. Procedure specific experience (with the technique of periosteal harvesting) was more important than the academic qualifications or years of surgical experience in general. These data must be considered when planning or interpreting the results of studies involving periosteal explantation or grafting, or when periosteum serves as a source of mesenchymal stem cells.     

The chondrogenic potential of periosteum decreases with age.

Periosteum contains undifferentiated mesenchymal stem cells that possess the potential for chondrogenesis during cartilage repair and in fracture healing. With aging, the chondrogenic potential of periosteum declines significantly. An organ-culture model was used to investigate the relationship between the chondrogenic potential of periosteum and aging. A total of 736 periosteal explants from the proximal medial tibiae of 82 rabbits, aged 2 weeks to 2 years, were cultured in agarose suspension conditions conductive for chondrogenesis. and analyzed using histomorphometry, collagen typing, wet weight measurement, 3H-thymidine and 35S-sulfate uptake, autoradiography, and PCNA immunostaining. The rabbits were skeletally mature by 6 months and stopped increasing in weight by 12 months. Chondrogenesis declined significantly with age (P < 0.0001) and was maximal in the 1.5-2 month-old rabbits. Explants from the 6 month-old rabbits formed 50% less cartilage. and by 12 months chondrogenesis reached a steady state minimal level. In parallel with this decrease in chondrogenic potential similar decreases were measured in 3H-thymidine uptake (P < 0.0001). 35S-sulfate uptake (P = 0.0117), as well as the thickness (P < 0.0001) and the total number of cells in the cambium layer of the periosteum (P < 0.0001). Autoradiography with 3H-thymidine and PCNA immunostaining confirmed the measured decrease in proliferative activity in the cambium layer where the chondrocyte precursors reside, although the percentage of proliferating cells did not change significantly with age. The most dramatic change was the marked decrease (87%) in the thickness and total cell number in the cambium layer of the perisoteum between the 2 and 12 month-old rabbits (P < 0.05). These data confirm a decline in the chondrogenic potential of periosteum with aging. Thus, one possibility for improving cartilage formation by periosteal transplantation after skeletal maturity would be to stimulate an increase in the total number of cells in the chondrocyte precursor pool early during chondrogenesis.     

Culturing periosteum in vitro: the influence of different sizes of explants

Periosteal transplantation is being used clinically to repair articular defects. Isolated cells and very small periosteal explants can be grown in tissue culture, but it will be necessary to test larger sizes for tissue engineering to be applied to clinical transplantation of periosteum. This study was conducted to assess the chondrogenic potential of different sizes of periosteal explants in agarose culture. Ninety-six rabbit tibial periosteal explants in three different sizes (small 1.5 × 2, medium 3 × 2, and large 4 × 6 mm, 32 pieces per size) were cultured in agarose suspension for 6 wk and given TGF-β1 (10 ng/mL) for the first 2 wk. Tissue growth, as indicated by normalized final wet weights of the explants after 6 wk in culture, was inversely proportional to explant size. Cartilage formation was observed in all explants. Histomorphometry revealed that cartilage formation was significantly better for the smaller explants (80% cartilage), but similar in the medium and larger explants (60% cartilage). Similar proportions of type II collagen were present in the different-sized explants. This study demonstrates that various sizes of periosteal explants can be grown in culture. Abundant cartilage was produced even by the large explants.     

Temporal expression patterns of BMP receptors and collagen II (B) during periosteal chondrogenesis.

Articular cartilage has a limited ability to repair itself. Periosteal grafts have chondrogenic potential and are used clinically to repair defects in articular cartilage. An organ culture model system for in vitro rabbit periosteal chondrogenesis has been established to study the molecular events of periosteal chondrogenesis in vitro. In this model, bone morphogenetic protein-2 (BMP2) mRNA expression was found to be upregulated in the first 12 h. BMPs usually transduce their signals through a receptor complex that includes type II and either type IA or type IB BMP receptors. Receptors IA and IB play distinct roles during limb development. We have examined the temporal expression patterns for the mRNAs of these receptors using our experimental model. The mRNA expression patterns of these three BMP receptors differed from one another in periosteal explants during chondrogenesis. When these explants were cultured under chondrogenic conditions (agarose suspension with TGF-beta1 added to the media for the first 2 days), the expression of BMPRII mRNA and that of BMPRIA mRNA varied only slightly and persisted over a long time. In contrast, the expression of BMPRIB mRNAwas upregulated within 12 h, peaked at day 5, and fell to a level that was barely detected beyond day 21. Moreover, the expression of BMPRIB mRNA preceded that of collagen type IIB mRNAs, a marker for matrix-depositing chondrocytes. These data support a role for coordinate expression of BMP2 and its receptors early during periosteal chondrogenesis.     

Relationship of donor site to chondrogenic potential of periosteum in vitro

Periosteum has been shown in vitro and in vivo to have a chondrogenic potential that permits it to be used for cartilage regeneration. A useful donor site should have good chondrogenic potential, availability of a large quantity of periosteum, and relative ease of access, and it should be associated with a low rate of morbidity. We hypothesized that the chondrogenic potential of periosteum varies from one bone to another and among different regions of the periosteum from a single bone. A total of 370 periosteal and 37 fascia lata (control) explants were taken from the skull, the ilium, the scapula, the upper, middle, and lower medial proximal tibia, the posterior proximal tibia, and the distal tibia of 2-month-old New Zealand rabbits. The explants were cultured for 6 weeks in agarose/Dulbecco's modified Eagle medium to which 10 ng/ml of transforming growth factor-β1 was added during the first 2 weeks. Skeletal muscle and fascia lata were used as controls. In addition, the thickness, cell density, and total cell count of the cambium layer were measured in 24 explants from the donor sites on the ilium and the upper, middle, and lower proximal tibia. At 6 weeks, histomorphometry and quantitative collagen typing were performed. The periosteal donor sites could be grouped into three categories according to chondrogenic potential: ilium (best), scapula and tibia, and skull (no chondrogenesis). The scapular periosteum was slightly better than that from the tibia. Within the tibia, the upper and middle zones of the proximal region were similar and were slightly better than the lower proximal tibia or the distal tibia. The cellularity of the cambium layer correlated positively with the amount of cartilage as a percentage of the total area. The results of this study indicate that iliac periosteum exhibited the best overall chondrogenic potential in vitro but that periosteum from the traditionally used medial proximal tibia also was excellent. Periosteum from the skull was not chondrogenic. The chondrogenic potential of periosteum varies from bone to bone and within the periosteum from one bone. This variation in chondrogenic potential among donor sites may be due to a difference in the total cell count of the cambium layer.     

Combined effects of insulin-like growth factor-1 and transforming growth factor-beta1 on periosteal mesenchymal cells during chondrogenesis in vitro

OBJECTIVE: Periosteum contains undifferentiated mesenchymal stem cells that have both chondrogenic and osteogenic potential, and has been used to repair articular cartilage defects. During this process, the role of growth factors that stimulate the periosteal mesenchymal cells toward chondrogenesis to regenerate articular cartilage and maintain its phenotype is not yet fully understood. In this study, we examined the effects of insulin-like growth factor-1 (IGF-1) and transforming growth factor-beta1 (TGF-beta1), alone and in combination, on periosteal chondrogenesis using an in vitro organ culture model. METHODS: Periosteal explants from the medial proximal tibia of 2-month-old rabbits were cultured in agarose under serum free conditions for up to 6 weeks. After culture the explants were weighed, assayed for cartilage production via Safranin O staining and histomorphometry, assessed for proliferation via proliferative cell nuclear antigen (PCNA) immunostaining, and assessed for type II collagen mRNA expression via in situ hybridization. RESULTS: IGF-1 significantly increased chondrogenesis in a dose-dependent manner when administered continuously throughout the culture period. Continuous IGF-1, in combination with TGF-beta1 for the first 2 days, further enhanced overall total cartilage growth. Immunohistochemistry for PCNA revealed that combining IGF-1 with TGF-beta1 gave the strongest proliferative stimulus early during chondrogenesis. In situ hybridization for type II collagen showed that continuous IGF-1 maintained type II collagen mRNA expression throughout the cambium layer from 2 to 6 weeks. CONCLUSION: The results of this study demonstrate that IGF-1 and TGF-beta1 can act in combination to regulate proliferation and differentiation of periosteal mesenchymal cells during chondrogenesis.     

Induction of CD-RAP mRNA during periosteal chondrogenesis.

Induction of chondrogenesis and maintenance of the chondrocyte phenotype are critical events for autologous periosteal transplantation, which is a viable approach for cartilage repair. Cartilage-derived retinoic acid-sensitive protein (CD-RAP) is a recently discovered protein that is mainly produced in cartilage. During development, CD-RAP expression starts at the beginning of chondrogenesis and continues throughout cartilage maturation. In order to investigate the involvement of CD-RAP during periosteal chondrogenesis we have determined the nucleotide sequence of the rabbit CD-RAP mRNA and utilized this information to evaluate the temporal and spatial expression pattern of CD-RAP at the mRNA level during chondrogenesis. When the periosteal explants were cultured under chondrogenic conditions, the expression of CD-RAP was induced, as shown by a 40-fold increase in CD-RAP mRNA between days 7 and 10. The temporal expression pattern of CD-RAP closely mimicked that of collagen type IIB mRNA. Also, the CD-RAP mRNA was localized to the matrix forming chondrocytes in the cambium layer of the periosteum by in situ hybridization as indicated by colocalization with collagen type II mRNA and positive safranin O staining. These data suggest a regulatory role of CD-RAP in periosteal chondrogenesis, which is potentially important for both cartilage repair and fracture healing via callus formation.     

自体骨膜游离移植的实验研究--不同龄动物大块关节软骨缺损修复的比较

目的研究比较自体骨膜移植软骨再生修复不同龄动物大块关节软骨缺损。方法用52只不同龄家兔自体骨膜游离移植修复大块关节软骨缺损,比较移植骨膜生发层朝向关节腔与松质骨时再生软骨的差别。结果经不同时期肉眼和组织学检查证实,幼年兔和成年兔的骨膜移植都能生成软骨,修复大块关节软骨缺损。在成年兔骨膜再生的软骨与成年兔本身周围正常软骨的厚度、组织结构一样。移植骨膜生发层朝向关节腔与松质骨二者间再生软骨结果无明显差别。结论骨膜具有再生软骨的能力,可用来移植修复关节软骨的缺损。骨膜移植生发层不同朝向对软骨再生无明显影响。成年后骨膜移植修复关节软骨缺损能够生成与自身相适应的软骨。     

Serum-free media for periosteal chondrogenesis in vitro.

Organ culture studies involving whole explants of periosteum have been useful for studying chondrogenesis, but to date the standard culture model for these explants has required the addition of fetal bovine serum to the media. Numerous investigators have succeeded in culturing chondrocytes and embryonic cells in serum-free conditions but there have been no studies focused on achieving a defined, serum-free media for culturing periosteal explants. The purpose of the present investigation was to determine if whole periosteal explants can be grown and produce cartilage in serum-free conditions, and to define the minimum media supplements that would be conducive to chondrogenesis. 321 periosteal explants were obtained from the medial proximal tibiae of 31 two month-old NZ white rabbits and cultured using a published agarose suspension organ culture model and DMEM for six weeks. The explants were cultured with and without fetal bovine serum or bovine serum albumin and exposed to transforming growth factor beta alone, a combination of growth factors we call ChondroMix (10 ng/ml transforming growth factor beta, 50 ng/ml basic fibroblast growth factor, and 5 microg/ml growth hormone), and/or ITS+ (2.08 microg/ml each of insulin, transferrin, and selenious acid, plus 1.78 microg/ml linoleic acid and 0.42 mg/ml BSA). Maximal chondrogenic stimulation in this study was observed with the combination of ChondroMix and ITS+. However, the minimal requirement to match or exceed the level of chondrogenic stimulation seen in the standard model (TGF-1 in 10% FBS) was achieved simply by the addition of 2.0 microg/ml insulin in 0.1% BSA-containing medium (p < 0.05). Therefore, based on our results, it would be reasonable to assume that insulin is the component in ITS+ responsible for the observed increase in total cartilage growth. Lower concentrations of insulin were not effective, suggesting that the observed effect of insulin requires activation of the IGF-1 receptor.     

Hydraulic elevation of the periosteum: a novel technique for periosteal harvest.

Periosteum has been promoted as a potential substrate for tissue engineering. Its principal virtues are that it has a source of pluripotential mesenchymal cells and chondrogenic growth factors located in the cambium layer, and it can serve as a template for directional evolution of neo-tissue. The clinical use and in vitro study of periosteum-derived neo-tissue has been limited by the level of surgical skill required for harvest. Precise surgical technique, task-specific experience, adequate volume of procedures, and general surgical expertise are required for optimal harvest using the traditional periosteal elevator method. This report describes an easily mastered technique that preserves viability while providing the harvest of relatively large amounts of periosteum. Skeletally mature New Zealand white rabbits (11 males/20 tibias; 4 females/8 tibias; approximate weight 3.5 kg) and one Yucatan miniature pig were used for harvest of periosteum from the tibia using the traditional periosteal elevator and the developed hydraulic elevation approach. Histologic examination of the periosteal explants obtained by the developed method showed preservation of the cambium layer containing the progenitor cells necessary for the generation of neo-cartilage. This technique provides a simple method of harvesting large segments (>5 cm x 1 cm) of periosteum in a single procedure and may facilitate better exploitation of periosteum in tissue engineering.     

The enhancement of periosteal chondrogenesis in organ culture by dynamic fluid pressure.

Cartilage repair by autologous periosteal arthroplasty is enhanced by continuous passive motion (CPM) of the joint after transplantation of the periosteal graft. However, the mechanisms by which CPM stimulate chondrogenesis are unknown. Based on the observation that an oscillating intra-synovial pressure fluctuation has been reported to occur during CPM (0.6-10 kPa), it was hypothesized that the oscillating pressure experienced by the periosteal graft as a result of CPM has a beneficial effect on the chondrogenic response of the graft. We have developed an in vitro model with which dynamic fluid pressures (DFP) that mimic those during CPM can be applied to periosteal explants while they are cultured in agarose gel suspension. In this study periosteal explants were treated with or without DFP during suspension culture in agarose, which is conducive to chondrogenesis. Different DFP application times (30 min, 4 h, 24 h/day) and pressure magnitudes (13, 103 kPa or stepwise 13 to 54 to 103 kPa) were compared for their effects on periosteal chondrogenesis. Low levels of DFP (13 kPa at 0.3 Hz) significantly enhanced chondrogenesis over controls (34 +/- 7% vs 14 +/- 5%; P < 0.05), while higher pressures (103 kPa at 0.3 Hz) completely inhibited chondrogenesis, as determined from the percentage of tissue that was determined to be cartilage by histomorphometry. Application of low levels of DFP to periosteal explants also resulted in significantly increased concentrations of Collagen Type II protein (43 +/- 8% vs 10 +/- 5%; P < 0.05). New proteoglycan synthesis, as measured by 35S-sulphate uptake was increased by 30% in periosteal explants stimulated with DFP (350 +/- 50 DPM vs 250 +/- 75 DPM of 35S-sulphate uptake/microg total protein), when compared to controls though this difference was not statistically significant. The DFP effect at low levels was dose-dependant for time of application as well, with 4 h/day stimulation causing significantly higher chondrogenesis than just 30 min/day (34 +/- 7 vs 12 +/- 4% cartilage; P < 0.05) and not significantly less than that obtained with 24 h/day of DFP (48 +/- 9% cartilage, P > 0.05). These observations may partially explain the beneficial effect on cartilage repair by CPM. They also validate an in vitro model permitting studies aimed at elucidating the mechanisms of action of mechanical factors regulating chondrogenesis. The fact that these tissues were successfully cultured in a mechanical environment for six weeks makes it possible to study the actions of mechanical factors on the entire chondrogenic pathway, from induction to maturation. Finally, these data support the theoretical predictions regarding the role of hydrostatic compression in fracture healing.     

Localization of chondrocyte precursors in periosteum

OBJECTIVE: Periosteal chondrogenesis is relevant to cartilage repair and fracture healing. Periosteum contains two distinct layers: a thick, outer fibrous layer and a thin, inner cambium layer which is adjacent to the bone. Specific chondrocyte precursors are known to exist in periosteum but have not yet been identified. In this study, the location of the chondrocyte precursors in periosteum was determined. METHOD: One hundred and twenty periosteal explants from 30 2-month-old NZ rabbits were cultured for up to 42 days. Histomorphological changes and spatio-temporal localization of Col. II mRNA and protein were analysed. RESULTS: On day 7, chondrocyte differentiation appeared in the most juxtaosseous region in the cambium layer. Col. II mRNA and protein were also evident in the same region. By day 14, chondrocyte differentiation progressed further into the juxtaosseous cambium layer, as did Col. II mRNA and protein. With growth of the neocartilage, the cambium layer gradually diminished to the extent that by 21-28 days it was no longer evident. Cartilage growth was significant and followed an appositional pattern, growing away from the fibrous layer. The fibrous layer remained essentially unchanged from 0-42 days, without evidence of hypertrophy or atrophy. Col. II mRNA expression was never seen in the fibrous layer. CONCLUSION: From these data, three conclusions can be drawn concerning chondrogenesis from periosteum: (1) the chondrocyte precursors are located in the cambium layer of periosteum; (2) chondrogenesis commences in the juxtaosseous area in the cambium layer and progresses from the juxtaosseous region to the juxtafibrous region of the cambium layer; (3) neocartilage growth is appositional, which displaces the fibrous layer away from the cartilage already formed, as new cartilage is formed between these two layers. These findings suggest that the least differentiated (stem or reserve) cells are located in the cambium layer furthest from the bone. CLINICAL RELEVANCE: These findings show that the chondrocyte precursors are located in the cambium layer of periosteum. Preservation of this layer is essential for chondrogenesis. As neocartilage growth is appositional, away from the fibrous layer, it can be expected that the new cartilage deposited in and adjacent to a periosteal graft would be expected to be located on the side of the cambium layer, rather than on the side of the fibrous layer of the graft.     

Transplantation of free tibial periosteal grafts for the repair of articular cartilage defect: An experimental study

Background:

Articular chondrocytes have got a long lifespan but rarely divides after maturity. Thus, an articular cartilage has a limited capacity for repair. Periosteal grafts have chondrogenic potential and have been used to repair defects in the articular cartilage. The purpose of the present study is to investigate the differentiation of free periosteal grafts in the patellofemoral joint where the cambium layer faces the subchondral bone and to investigate the applicability of periosteal grafts in the reconstruction of articular surfaces.

Materials and Methods:

The study was carried out over a period of 1 year on 25 adult, male Indian rabbits after obtaining permission from the institutional animal ethical committee. A full-thickness osteochondral defect was created by shaving off the whole articular cartilage of the patella of the left knee. The defect thus created was grafted with free periosteal graft. The patella of the right knee was taken as a control where no grafting was done after shaving off the articular cartilage. The first animal was used to study the normal histology of the patellar articular cartilage and periosteum obtained from the medial surface of tibial condyle. Rest 24 animals were subjected to patellectomy, 4 each at serial intervals of 2, 4, 8, 16, 32 and 48 weeks and the patellar articular surfaces were examined macroscopically and histologically.

Results:

The grafts got adherent to the underlying patellar articular surface at the end of 4 weeks. Microscopically, graft incorporation could be appreciated at 4 weeks. Mesenchymal cells of the cambium layer were seen differentiating into chondrocytes by the end of 4 weeks in four grafts (100%) and they were arranged in a haphazard manner. Till the end of 8 weeks, the cellular arrangement was mostly wooly. At 16 weeks, one graft (25%) had wooly arrangement of chondrocytes and three grafts (75%) had columnar formation of cells. Same percentage was maintained at 32 weeks. Four grafts (100%) at 48 weeks showed columnar orientation. The control side showed no changes over the shaved off articular surface in all the rabbits. One rabbit at 4 weeks had a dislocation of the patella on the control side. None of the rabbits developed any infection or wound dehiscence.

Conclusion:

Autologous periosteal graft transplantation can be a promising substitute for articular cartilaginous defects.     

Clonal growth of human articular cartilage and the functional role of the periosteum in chondrogenesis

OBJECTIVE: Clinical cartilage repair with transplantation of cultured chondrocytes, the first described technique introduced in 1994, includes a periosteal membrane but today cells are also implanted without the periosteal combination. The aim of this study was to see if the periosteum had more than a biomechanical function and if the periosteum had a biological effect on the seeded cells tested in an agarose system in which the clonal growth in agarose and the external growth stimulation could be analysed. METHODS: Four different experiments were used to study the growth of human chondrocytes in agarose and the periosteal influence. Human chondrocytes were isolated and transferred to either primary or secondary agarose culture. After 4 weeks, the total number of clones >50 microm was counted. Cocultures of chondrocytes and periosteal tissue, cultures of chondrocytes with conditioned medium from chondrocytes, periosteal cells and fibroblast were used to study a potential stimulatory effect on growth and different cytokines and growth factors were analysed. RESULTS: It was found that the human chondrocytes had different growth properties in agarose with the formation of four different types of clones: a homogenous clone without matrix production, a homogenous clone with matrix production, a differentiated clone with matrix production and finally a differentiated clone without matrix production. The periosteum exerted a paracrine effect on cultured chondrocytes in agarose resulting in a higher degree of cloning. The chondrocytes produced significant amounts of interleukin (IL)-6, IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF) and transforming growth factor (TGF)-beta. The periosteum produced significant amounts of IL-6, IL-8 and TGF-beta. Cocultures of chondrocytes and periosteum demonstrated a potentiation of IL-6 and IL-8 release but not of TGF-beta and GM-CSF. CONCLUSION: Articular chondrocytes are able to form clones of different properties in agarose and the periosteum has a capacity of stimulating chondrocyte clonal growth and differentiation and secretes significant amounts of IL-6, IL-8, GM-CSF and TGF-beta. It may be that the repair of cartilage defects with seeded chondrocytes could benefit from the combination with a periosteal graft. The production of TGF-beta by implanted chondrocytes could influence the chondrogenic cells in the periosteum to start a periosteal chondrogenesis and together with the matrix from implanted chondrocyte production, a repair of cartilaginous appearance may develop; a dual chondrogenic response is possible.     

Role of oxygen tension during cartilage formation by periosteum

Tissue engineering makes regeneration of cartilage possible but requires optimization of culture conditions. The effects of oxygen tension on cartilage metabolism are controversial in the literature, and we could find no information detailing the optimal oxygen concentration for growing new cartilage (neochondrogenesis). Periosteal cells and tissues can be used to grow cartilage in vivo and in vitro. In this study, using a standard periosteal organ culture model, we found that cartilage formation by periosteal explants is affected by the ambient oxygen concentrations. A total of 480 periosteal explants from 30 2-month-old New Zealand White rabbits were cultured in agarose suspension at different oxygen concentrations (1-90%) for 6 weeks. Chondrogenesis, which was analyzed by histomorphometry and quantitative collagen typing, was maximal at 12–15% oxygen. There were no significant differences in chondrogenesis in the range of 12–45%. There was inhibition of cartilage and type II collagen formation at very high (90%) and very low (1–5%) oxygen concentrations. However, contrary to what some have thought, chondrogenesis is maximal under aerobic conditions. If this is true for systems other than periosteal implants, it would have important implications for growing cartilage in vitro.     

The cellular origin of cartilage-like tissue after periosteal transplantation of full-thickness articular cartilage defects: an experimental study using transgenic rats expressing green fluorescent protein

BACKGROUND: Periosteal transplantation is commonly used for the treatment of articular cartilage defects. However, the cellular origin of the regenerated tissue after periosteal transplantation has not been well defined. The objective of this study was to investigate the cellular origin of the regenerated tissue after periosteal transplantation. METHOD: Free periosteum was harvested from the tibia of 10-week-old adolescent enhanced green fluorescent protein (GFP-) expressing transgenic Sprague Dawley (SD) rats and was transplanted to full-thickness articular cartilage defects of the patellar groove in normal 10-week-old adolescent SD rats. The periosteum was sutured to the defect with the cambium layer facing the joint cavity. 8 SD rats were killed at 4 weeks and 8 SD rats were killed at 8 weeks after surgery. The repaired tissue was assessed histologically and histochemically. GFP-positive cells derived from the donor periosteum could easily be detected in the repaired tissue by use of a fluorescent microscope. RESULTS: At both 4 and 8 weeks after transplantation, the entire area of the defects had been repaired, with the regenerated tissue being well stained histologically with safranin-O. Most cells in the whole area of the regenerated tissue were GFP-positive, indicating that very few of the cells were GFP-negative cells originating from the recipient rats. INTERPRETATION: This experiment demonstrates that most cells in regenerated tissue after periosteal transplantation using adolescent animals do not originate from recipient cells but from the periosteal cells of the donor.     

Reconstruction of Articular Cartilage Defects with Free Periosteal Grafts: An Experimental Study

The chondrogenic potential of free autogenous periosteal grafts was studied histologically in 6-month-old rabbits. The grafts were taken from the tibia and transplanted to 7 × 14 mm large artificial defects of the femoral articular cartilage. The results revealed that the defects were repaired and filled after 4 weeks with a hyaline-like cartilage which was histologically similar to the cartilage adjacent to the transplant. The tissue maintained this morphology after 1 year of observation. In control animals where no periosteum was transplanted to the defect, no real cartilage was found. The tissue which partially filled the defect was a variable mixture of fibrous tissue and fibrocartilage.     

Brief exposure to high-dose transforming growth factor-beta1 enhances periosteal chondrogenesis in vitro: a preliminary report

BACKGROUND: Articular cartilage has limited potential for repair. There have been various attempts aimed at improving the repair process in articular cartilage. Transforming growth factor-beta1 (TGF-beta1) has a stimulatory effect on chondrogenesis in periosteal explants. The purpose of the present study was to determine the effect of brief exposures (i.e., thirty and sixty minutes) of high concentrations of TGF-beta1 on periosteal chondrogenesis. METHODS: Five hundred and seventy-three periosteal explants were harvested from forty-six two-month-old male New Zealand White rabbits. Explants were exposed to 50 or 100 ng/mL of TGF-beta1 for thirty or sixty minutes. The amount of cartilage formed was then determined with use of a standardized six-week agarose culture assay. RESULTS: There was a significant increase in the amount of cartilage formation (p < 0.01), Type-II collagen content (p < 0.05), and sulfate incorporation (p < 0.0001) in explants treated with TGF-beta1. Maximal stimulation occurred following exposure to 100 ng/mL of TGF-beta1 for thirty minutes. There was also an increase in chondrocyte proliferation as measured by [ (3) H-] thymidine incorporation on day 5 of culture (p < 0.049). Conclusions: The findings of this study indicate that exposure to TGF-beta1 has a stimulatory effect on periosteal chondrogenesis. This stimulatory effect is observed even with a very brief exposure time of thirty minutes. Clinical Relevance: A possible clinical application of these findings is exposure of periosteal grafts that are currently utilized clinically to resurface articular defects to TGF-beta1 during the short time between graft procurement and implantation into the joint. This may obviate the need for intra-articular administration of TGF-beta1 and may enhance the ultimate graft incorporation and quality of cartilage repair.     

The cellular origin of cartilage-like tissue after periosteal transplantation of full-thickness articular cartilage defects: An experimental study using transgenic rats expressing green fluorescent protein

Background?Periosteal transplantation is commonly used for the treatment of articular cartilage defects. However, the cellular origin of the regenerated tissue after periosteal transplantation has not been well defined. The objective of this study was to investigate the cellular origin of the regenerated tissue after periosteal transplantation.

Method?Free periosteum was harvested from the tibia of 10-week-old adolescent enhanced green fluorescent protein (GFP-) expressing transgenic Sprague Dawley (SD) rats and was transplanted to full-thickness articular cartilage defects of the patellar groove in normal 10-week-old adolescent SD rats. The periosteum was sutured to the defect with the cambium layer facing the joint cavity. 8 SD rats were killed at 4 weeks and 8 SD rats were killed at 8 weeks after surgery. The repaired tissue was assessed histologically and histochemically. GFP-positive cells derived from the donor periosteum could easily be detected in the repaired tissue by use of a fluorescent microscope.

Results?At both 4 and 8 weeks after transplantation, the entire area of the defects had been repaired, with the regenerated tissue being well stained histologically with safranin-O. Most cells in the whole area of the regenerated tissue were GFP-positive, indicating that very few of the cells were GFP-negative cells originating from the recipient rats.

Interpretation?This experiment demonstrates that most cells in regenerated tissue after periosteal transplantation using adolescent animals do not originate from recipient cells but from the periosteal cells of the donor.

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