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.     

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.     

Periosteum responds to dynamic fluid pressure by proliferating in vitro.

Periosteum provides a source of undifferentiated chondrocyte precursor cells for fracture healing that can also be used for cartilage repair. The quantity of cartilage that can be produced, which is a determining factor in fracture healing and cartilage repair, is related to the number of available stem cells in the cambium layer. Cartilage formation during both of these processes is enhanced by motion of the fracture or joint in which periosteum has been transplanted. The effect of dynamic fluid pressure on cell proliferation in periosteal tissue cultures was determined in 452 explants from 60 immature (2-month-old) New Zealand White rabbits. The explants were cultured in agarose suspension for 1-14 days. One group was subjected to cyclic hydrostatic pressure, which is referred to as dynamic fluid pressure, at 13 kPa and a frequency of 0.3 Hz. Control explants were cultured in similar chambers without application of pressure. DNA synthesis ([3H]thymidine uptake) and total DNA were measured. The temporal pattern and distribution of cell proliferation in periosteum were evaluated with autoradiography and immunostaining with proliferating cell nuclear antigen. Dynamic fluid pressure increased proliferation of periosteal cells significantly, as indicated by a significant increase in [3H]thymidine uptake at all time points and a higher amount of total DNA compared with control values. On day 3, when DNA synthesis reached a peak in periosteal explants, [3H]thymidine uptake was 97,000+/-5,700 dpm/microg DNA in the group exposed to dynamic fluid pressure and 46,000+/-6,000 dpm/microg in the controls (p < 0.001). Aphidicolin, which blocks DNA polymerase alpha, inhibited [3H]thymidine uptake in a dose-dependent manner in the group subjected to dynamic fluid pressure as well as in the positive control (treated with 10 ng/ml of transforming growth factor-beta1) and negative control (no added growth factors) groups, confirming that [3H]thymidine measurements represent proliferation and dynamic fluid pressure stimulates DNA synthesis. Total DNA was also significantly higher in the group exposed to dynamic fluid pressure (5,700+/-720 ng/mg wet weight) than in the controls (3,700+/-630) on day 3 (p < 0.01). Autoradiographs with [3H]thymidine revealed that one or two cell cycles of proliferation took place in the fibrous layer prior to proliferation in the cambium layer (where chondrocyte precursors reside). Proliferating cell nuclear antigen immunophotomicrographs confirmed the increased proliferative activity due to dynamic fluid pressure. These findings suggest either a paracrine signaling mechanism between the cells in these two layers of the periosteum or recruitment/migration of proliferating cells from the fibrous to the cambium layer. On the basis of the data presented in this study, we postulate that cells in the fibrous layer respond initially to mechanical stimulation by releasing growth factors that induce undifferentiated cells in the cambium layer to divide and differentiate into chondrocytes. These data indicate that cell proliferation in the early stages of chondrogenesis is stimulated by mechanical factors. These findings are important because they provide a possible explanation for the increase in cartilage repair tissue seen in joints subjected to continuous passive motion. The model of in vitro periosteal chondrogenesis under dynamic fluid pressure is valuable for studying the mechanisms by which mechanical factors might be involved in the formation of cartilage in the early fracture callus and during cartilage repair.     

Organization and cellular biology of the perichondrial ossification groove of ranvier: a morphological study in rabbits.

The perichondrial ossification groove of Ranvier, a circumferential groove in the periphery of the epiphyseal cartilage, was studied in rabbits whose ages ranged from one week to eight months using light and electron microscopy, autoradiography after labeling with 3H-thymidine, 3H-proline, and 3H-glucosamine, and histochemical staining for proteoglycans and alkaline phosphatase. By these methods, three groups of cells were identified within the groove: 1. A group of densely packed cells deep in the groove, which are the progenitor cells for the osteoblasts that form the bone bark, a cuff of bone surrounding the epiphyseal growth-plate region and the adjacent part of the metaphysis. 2. A group of more widely dispersed, relatively undifferentiated mesenchymal cells and fibroblasts, some of which are chondroblast precursors that probably contribute to appositional chondrogenesis and growth in width of the epiphyseal cartilage. 3. Fibroblasts and fibrocytes among sheets of highly oriented and organized collagen fibers which form a fibrous layer that is continuous with the outer fibrous layer of the periosteum and with the perichondrium. This layer also sends fibers into the epiphyseal cartilage and anchors the periosteum firmly to the epiphyses as bone growth proceeds.     

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.     

Behaviour of cancellous bone graft with and without periosteal isolation in striated muscle. An experimental study

The capacity of the periosteum to inhibit resorption of cancellous bone grafts into muscle was investigated in 34 four- to six-week-old rabbits. In 17 experiments the periosteum was wrapped around the grafts with the cambium layer facing the bone, and in seven experiments with the cambium layer facing the muscle. In the control group of 10 experiments there was no periosteal wrapping around the bone grafts. In Series 1 with the cambium layer of the periosteum facing the bone, after 20 weeks a tubular bone with Haversian system and bone marrow was seen. The transplants were surrounded by normal-looking periosteum. Bone formation from the periosteum occurred through enchondral ossification. Inductive bone growth was observed from the cancellous graft. In Series 2 with the cambium layer facing the surrounding muscle tissue, after 20 weeks two laminar bone blocks with periosteum in between and surrounding each block was observed. In the control series without periosteal covering, after 20 weeks only fibrous tissue remained in the transplantation site. It is obvious that periosteal isolation of cancellous bone grafts inhibits their resorption when transplanted into muscle in young animals.     

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.     

Transforming growth factor beta 1 stimulates type II collagen expression in cultured periosteum-derived cells.

Chondrogenesis can occur during a bone repair process, which is related to several growth factors. Transforming growth factor beta 1 (TGF-beta 1) downregulates the expression of type II collagen by chondrocytes in vitro, but injection of TGF-beta 1 into the periosteum in vivo increases type II collagen mRNA levels and initiates chondrogenesis. We examined the effect of TGF-beta 1 on collagen gene expression in a bovine periosteum-derived cell culture system to evaluate its direct effect on the periosteum. Cultured cells expressed alkaline phosphatase and collagen pro alpha 1(I) and pro alpha 1(II) mRNAs. A low level of type II collagen synthesis was demonstrated by immunoprecipitation. TGF-beta 1 had no effect on periosteal cell proliferation. Expression of collagen pro alpha 1(I) mRNA did not change with TGF-beta 1 treatment, but alkaline phosphatase mRNA showed a dose-dependent decrease. Expression of collagen pro alpha 1(II) mRNA was stimulated 2.7-fold by TGF-beta 1. TGF-beta 1 also caused a 2.6-fold increase in type II collagen synthesis by immunoprecipitation. These findings indicate that TGF-beta 1 is an enhancer of the expression of the chondrocyte phenotype of the periosteal cells and suggest that TGF-beta 1 is important in initiating and promoting cartilage formation in vivo.     

Cambium cell stimulation from surgical release of the periosteum.

An autograft of periosteal tissue containing cambium cells has potential to become chondrogenic or osteogenic depending on the regeneration repair strategies. The potential number of harvestable cambium cells diminishes with age. Other factors may be associated with a reduction in the number or variable yields of cambium cells including harvest technique, harvest site location, and the time interval from harvest to implantation. Attempts to increase the number of cambium cells have included improvements in harvesting and handling technique, and expansion of the cells in tissue culture. An "in situ" stimulation and proliferation technique would offer the potential for increasing the number of cambium cells in a cost-effective manner for transplantation without the need for expansion in tissue culture.The hypothesis tested was that surgical release of the periosteum and its deep inner underlying cambium layer by sharply incising through the superficial periosteal fibrous layer down to and scoring the cortical bone surface would increase the number of cambium cells that could be harvested at a later time period. Two techniques for periosteal release were used to stimulate a proliferation of the underlying cambium layer and increase the cambium cells for harvest in skeletally mature goats: (1) sharply scoring all four-sides of the tissue test site perimeter, and (2) sharply scoring only two sides of the tissue test site.The two-sided and four-sided release scoring of the periosteum induced stimulatory responses in the number of cambium cells. In addition, a marked increase in mRNA expression for BMP-2 (p<0.001) was observed within 24 h and remained elevated over baseline values for up to 96 h after this stimulation to the cambium layer.     

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。     

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.     

The periosteum. Part 1: Anatomy, histology and molecular biology

The periosteum is a thin layer of connective tissue that covers the outer surface of a bone in all places except at joints (which are protected by articular cartilage). As opposed to bone itself, it has nociceptive nerve endings, making it very sensitive to manipulation. It also provides nourishment in the form of blood supply to the bone. The periosteum is connected to the bone by strong collagenous fibres called Sharpey's fibres, which extend to the outer circumferential and interstitial lamellae of bone. The periosteum consists of an outer "fibrous layer" and inner "cambium layer". The fibrous layer contains fibroblasts while the cambium layer contains progenitor cells which develop into osteoblasts that are responsible for increasing bone width. After a bone fracture the progenitor cells develop into osteoblasts and chondroblasts which are essential to the healing process. This review discusses the anatomy, histology and molecular biology of the periosteum in detail.     

Reconstruction of patellar cartilage defects with free periosteal grafts. An experimental study

The osteo-chondrogenic potential of free periosteal grafts was investigated within the knee joint in 26 rabbits aged four to six weeks. A total of 36 knee joints were operated on. The grafts were stripped from the medial side of the right tibia and sutured on the articular surface of the patella, from which the cartilage had been totally excised to the subchondral bone. In 16 knees the graft was sutured with the cambium layer towards the subchondral bone and in eight knees the fibrous layer faced the bone. In the control group of twelve knees the patellar articular cartilages were excised and no periosteal transplant was grafted to the patellar articular surface. In the transplantation group cartilage formation could be seen already one week after the operation. There were no marked differences between the series with the cambium layer facing the subchondral bone or the group with the fibrous layer facing the bone. At 20 weeks the hypertrophied cartilage had thinned and resembled normal joint cartilage. In the control group the histological picture resembled osteoarthritis.     

Differential phenotypic characteristics of heterogeneous cell population in the rabbit periosteum

BACKGROUND: Periosteum and periosteum-derived progenitor cells have demonstrated the potential for stimulative applications in repair of various musculoskeletal tissues. It has been found that the periosteum contains mesenchymal progenitor cells that are capable of differentiating into either osteoblasts or chondrocytes, depending on the culture conditions. Anatomically, the periosteum is a heterogeneous multilayered membrane, consisting of an outer fibrous and an inner cambium layer. The present study was designed to elucidate the phenotypic characteristics of fibrous and cambium layer cells in vitro. METHODS: Using a sequential enzymatic digestion method, fibrous and cambium layer cells were harvested separately from periosteum-bone explants of the proximal tibia of 6-month-old New Zealand White rabbits. RESULTS: We found that the cells from each layer showed distinct phenotypic characteristics in a primary monolayer culture system. Specifically, the cambium cells demonstrated higher osteogenic characteristics (higher alkaline phosphatase and osteocalcin levels) than the fibrous cells. However, these differences diminished with time in vitro. INTERPRETATION: Our findings suggest that the periosteum has phenotypically distinct heterogeneous cell populations. Care must be taken in order to identify and distinguish the intrinsic phenotypes of the heterogeneous periosteum-derived cell types 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.     

Initial characterization of PTH-related protein gene-driven lacZ expression in the mouse.

The PTHrP gene generates low-abundance mRNA and protein products that are not easily localized by in situ hybridization histochemistry or immunohistochemistry. We report here a PTHrP-lacZ knockin mouse in which beta-gal activity seems to provide a simple and sensitive read-out of PTHrP gene expression. INTRODUCTION: PTH-related protein (PTHrP) is widely expressed in fetal and adult tissues, typically as low-abundance mRNA and protein products that maybe difficult to localize by conventional methods. We created a PTHrP-lacZ knockin mouse as a means of surveying PTHrP gene expression in general and of identifying previously unrecognized sites of PTHrP expression. MATERIALS AND METHODS: We created a lacZ reporter construct under the control of endogenous PTHrP gene regulatory sequences. The AU-rich instability sequences in the PTHrP 3' untranslated region (UTR) were replaced with SV40 sequences, generating products with lacZ/beta gal kinetics rather than those of PTHrP. A nuclear localization sequence was not present in the construct. RESULTS: We characterized beta-galactosidase (beta-gal) activity in embryonic whole mounts and in the skeleton in young and adult animals. In embryos, we confirmed widespread PTHrP expression in many known sites and in several novel epidermal appendages (nail beds and footpads). In costal cartilage, beta-gal activity localized to the perichondrium but not the underlying chondrocytes. In the cartilaginous molds of forming long bones, beta-gal activity was first evident at the proximal and distal ends. Shortly after birth, the developing secondary ossification center formed in the center of this PTHrP-rich chondrocyte population. As the secondary ossification center developed, it segregated this population into two distinct PTHrP beta-gal+ subpopulations: a subarticular subpopulation immediately subjacent to articular chondrocytes and a proliferative chondrocyte subpopulation proximal to the chondrocyte columns in the growth plate. These discrete populations remained into adulthood. beta-gal activity was not identified in osteoblasts but was present in many periosteal sites. These included simple periosteum as well as fibrous tendon insertion sites of the so-called bony and periosteal types; the beta-gal-expressing cells in these sites were in the outer fibrous layer of the periosteum or its apparent equivalents at tendon insertion sites. Homozygous PTHrP-lacZ knockin mice had the expected chondrodysplastic phenotype and a much expanded region of proximal beta-gal activity in long bones, which appeared to reflect in large part the effects of feedback signaling by Indian hedgehog on proximal cell proliferation and PTHrP gene expression. CONCLUSIONS: The PTHrP-lacZ mouse seems to provide a sensitive reporter system that may prove useful as a means of studying PTHrP gene expression.     

Repair of full-thickness cartilage defects in rabbit knees with free periosteal graft preincubated with transforming growth factor

This study compared different concentrations of transforming growth factor-beta 1 (TGF-beta1) used for short-term preincubation in vitro of large periosteal explants to determine the effect of chondrogenesis and the fate of repair cartilage over time. Eighty-two rabbits were divided into four groups: group A, non-grafted; group B, non-incubated grafted; group C, 100 ng/mL recombinant human (rh) TGF-beta1 preincubated grafted; and group D, 20 ng/mL rhTGF-beta1 preincubated grafted. Rabbits from each group were sacrificed at intervals between 2 and 24 weeks. Histologic slides were stained with safranin O and were scored based on a subjective scoring system. Group A healed with non-cartilaginous material. Group B healed with hyaline cartilage-like material with progressive thinning of this regenerated layer; at 24 weeks, this layer was fibrous tissue. Group C enhanced repair with hyaline cartilage-like material but accelerated early degeneration and osteophyte formation; the cartilage became fibrous tissue at 24 weeks. Group D did not enhance cartilaginous repair. At 24 weeks, all groups had the same result. The 100 ng/mL rhTGF-beta1 preincubation in vitro with periosteum enhanced early osteochondral repair but did not show prolonged durability. Clinical application of this growth factor necessitates further study.     

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.