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Influence of platelet time activation on articular cartilage growth in the rabbit knee. Preliminary study
Abstract
The aim of the present article was to study the influence of platelets and different time activation on cartilage growth in articular defects in the rabbit knee.
Twelve male New Zealand rabbits (12 weeks) were divided in two groups. Under general anaesthesia, a 4 mm diameter and 2 mm deep defect was performed in medial condyles in both knees. The right knee defect was filled with platelet concentrate 5 min after being activated with ClCa in group A, and 2 min afterwards in group B. Platelets were obtained by centrifuging 10 ml arterial blood from the rabbit prior to the surgical procedure. The left knee defect was not filled. Rabbits were sacrificed 6 weeks after surgery. Macroscopic and microscopic studies were performed.
In group A, hyaline cartilage was observed in the right knee defect at the end of the experiment in five rabbits. None of the defects of the left knees showed hyaline cartilage growth. In group B, hyaline cartilage was observed in the right knee defect in only one rabbit. Nevertheless, in group B, all rabbits presented better chondral cellularity and regeneration and lower fibrosis in defects treated with platelets than in non-treated ones.
This technique for articular defect reconstruction with platelets is simple and easy, and has shown satisfactory results in our study. Platelets may be useful as an autologous source of multiple growth factors for articular defect reconstruction. Nevertheless, this is a preliminary study and further research is required.
... In the literature review (Table 1), a total of 31 papers 7-37 were found to meet the above-noted criteria. Of these, the vast majority (28 of 31, > 90%) [7][8][9][10][11]13,[15][16][17][18][19][20][21][22][23][24][25][26][27][28][30][31][32][33][34][35][36][37] utilized anterior approaches to access the MFC surface. More than half (17 of 28) of these anterior approach studies 7,8,10,11,[16][17][18][19]21,23,25,27,[32][33][34]36,37 regarded the site of the experimental insult as being in the weight-bearing region. ...
... More than half (17 of 28) of these anterior approach studies 7,8,10,11,[16][17][18][19]21,23,25,27,[32][33][34]36,37 regarded the site of the experimental insult as being in the weight-bearing region. The insult site through anterior approach (when identified in figures) was located in the cranial half region of the MFC surface ( Figure 2B) in all cases [7][8][9][10][11]13,[15][16][17][18][19][20][21][22][23][24][26][27][28]30,[32][33][34][35][36] . Posterior approaches were utilized in only three studies 12,14,29 , one of which was from our group. ...
This article addresses considerations for using a posterior (popliteal) instead of anterior (para-patellar) approach for experimental insult to the rabbit knee medial femoral condyle (MFC) surface in vivo. The posterior approach is particularly advantageous when intending to address the pathomechanisms of OA associated with habitual cartilage loading, or the efficacy of a cartilage repair method, in a clinically relevant experimental setting.
Studies using anterior versus posterior approaches for such purposes in survival rabbit models of the MFC articular surface insults were systematically surveyed. The anterior-posterior span of the primary weight-bearing region of that surface was demonstrated cadaverically.
Of a total of 31 papers identified in 2007-2012, an anterior approach was utilized in 28 studies (> 90%). More than half (17/28) explicitly regarded the cranial half (inferior aspect) of the MFC surface as being a "weight-bearing" region. The insult site through anterior approach (identified in figures) was located in the cranial half region in all cases. Cadaverically, however, the center of habitual tibio-femoral contact locations on the MFC surface was located in the caudal half region (posterior aspect) of the MFC surface. The majority of the habitual contact region was accessible only by a posterior surgical approach.
For the above-noted purposes, use of a posterior (popliteal) approach, rather than an anterior approach, is highly recommended.
... PPAR signaling consists of a group of nuclear receptor proteins, which functions as transcription factors to regulate cartilage growth and development [27]. Platelet activation serves as a key process during cartilage repair, since injured cartilage with platelet activation presents better chondral cellularity and regeneration [28]. Focal adhesion is the contact point that attaches chondrocytes to the pericellular cartilage matrix and links to intracellular organelles via cytoskeleton, and is involved in multiple cellular activities such as migration, proliferation, and gene expression [29]. ...
Background
Deer antler is a zoological exception due to its fantastic characteristics, including amazing growth rate and repeatable regeneration. Deer antler has been used as a key ingredient in traditional Chinese medicine relating to kidney and bone health for centuries. The aim of this study was to dissect the molecular regulation of deer antler extract (DAE) on xiphoid cartilage (XC).
Methods
The DAE used in this experiment was same as the one that was prepared as previously described. The specific pathogen-free (SPF) grade Sprague-Dawley (SD) rats were randomly divided into blank group ( n =10) and DAE group ( n =10) after 1-week adaptive feeding. The DAE used in this experiment was same as the one that was prepared as previously described. The rats in DAE group were fed with DAE for 3 weeks at a dose of 0.2 g/kg per day according to the body surface area normalization method, and the rats in blank group were fed with drinking water. Total RNA was extracted from XC located in the most distal edge of the sternum. Illumina RNA sequencing (RNA-seq) in combination with quantitative real-time polymerase chain reaction (qRT-PCR) validation assay was carried out to dissect the molecular regulation of DAE on XC.
Results
We demonstrated that DAE significantly increased the expression levels of DEGs involved in cartilage growth and regeneration, but decreased the expression levels of DEGs involved in inflammation, and mildly increased the expression levels of DEGs involved in chondrogenesis and chondrocyte proliferation.
Conclusions
Our findings suggest that DAE might serve as a complementary therapeutic regent for cartilage growth and regeneration to treat cartilage degenerative disease, such as osteoarthritis.
... The repair tissue produced at the site of cartilage defects treated with L-PRP implantation was both macroscopically and histologically superior in comparison to untreated defects at all sampling times, although complete characteristics of normal hyaline articular cartilage were not achieved at the end of the 24 weeks of study period. Our results are in accordance with earlier in vivo studies indicating the positive effect of PRP on articular cartilage repair and regeneration (23)(24)(25). Similar to our study, the platelet Fibrous tissue with numerous blood vessels and fibroblasts and cystic cavities can be seen at 4 weeks in all the treatment groups. ...
Background: Articular cartilage injuries of the knee are among the most debilitating injuries leading to osteoarthritis due to limited
regenerative capability of cartilaginous tissue. The use of platelet concentrates containing necessary growth factors for cartilage healing
has recently emerged as a new treatment method.
Objectives: The efficacy of two types of different platelet concentrates were compared in the treatment of acute articular cartilage injuries
of the knee in an animal model.
Materials and Methods: Eighteen adult Iranian mixed breed male dogs were used to conduct this experimental study. Full thickness
articular cartilage defects (diameter 6 mm, depth 5 mm) were created in the weight bearing area of femoral condyles of both hind limbs in
all dogs (n = 72). Twelve dogs were randomly selected to receive treatment and their right and left hind limb defects were treated by L-PRP
and L-PRF implantation respectively, while no treatment was undertaken in six other dogs as controls. The animals were euthanized at 4,
16 and 24 weeks following surgery and the resultant repair tissue was investigated macroscopically and microscopically. At each sampling
time, 4 treated dogs and 2 control dogs were euthanized, therefore 8 defects per group were evaluated.
Results: Mean macroscopic scores of the treated defects were higher than the controls at all sampling times with significant differences (P
< 0.05) observed between L-PRF treated and control defects (10.13 vs. 8.37) and L-PRP treated and control defects (10 vs. 8.5) at 4 and 16 weeks,
respectively. A similar trend in mean total microscopic scores was observed with a significant difference (P < 0.05) between L-PRP treated
and control defects at 4 (9.87 vs. 7.62) and 16 (13.38 vs. 11) weeks. No significant difference was observed between the platelet concentrate
treated defects in either mean macroscopic scores or mean total microscopic scores.
Conclusions: Both L-PRP and L-PRF could be used to effectively promote the healing of articular cartilage defects of the knee.
Keywords: Platelet-Rich Plasma; Cartilage; Knee Joint; Dogs; Articular Cartilage
... Articular cartilage is an avascular tissue, and partial thickness defects of the articular cartilage have poor healing potential (Calvo et al. 2004; Ytrehus et al. 2007 ). Conditions can vary in severity depending upon the location and extent of degeneration of cartilage within the knee; the treatment of articular cartilage injuries remains a challenge because cartilage have a limited capacity for spontaneous repair (García-Alvarez et al. 2008). The most common cause of a cartilage defect in the knee is trauma and is commonly seen in association with ligament injuries such as ACL tears (Hayashi et al. 2004; Gianotti et al. 2009). ...
Background: Knee articular cartilage full-thickness defects have a poor healing capacity and may progress to osteoarthritis and the need for knee replacement. This study was performed to determine the healing effect of BioGlue in medial femoral condyle articular cartilage full-thickness defects using the rabbit as an animal model. Materials and Methods: Forty-eight male rabbits were randomly divided into four equal groups (A-D). In group A, 4-mm articular cartilage defects were created in the right and left medial femoral condyles. A graft from the xiphoid cartilage was transplanted into the defect together with a designed BioGlue and the knees were closed. In group B, the articular cartilage defect was created in the same way as in group A, but the defect size was 6 mm. In group C, 4- and 6-mm articular cartilage defects were created in the right and left medial femoral condyles, respectively. The graft was transplanted into the defects and the knees were sutured. In group D, articular cartilage defects were created similar to those in group C, but filled with BioGlue and closed. The rabbits were euthanized and sub-grouped as A1 to D1 after 30 days and A2 to D2 after 60 days. Cartilage samples from the different experimental groups were examined macroscopi-cally and histologically for any differences between the groups. Results: Microscopic and macroscopic observations showed that BioGlue had a significant healing effect in the femoral condyle. Conclusions: Addition of BioGlue alone can effectively promote the healing of articular cartilage defects.
Articular cartilage defect is a condition leading to osteoarthritis of the joints if they remain unhealed or untreated. Some available surgical adhesives are used to adhere the wandering cartilaginous particle and avoid further joint damage. Healing as well as adhering properties of different substances has always been a topic of concern. Magnetic resonance imaging (MRI) and computed tomography (CT) are used to diagnose
the condition. This is an effort to evaluate the healing and adhering properties of a newly invented bioglue, both with MRI and CT. Sixty adult, Dutch, male rabbits used for this study were divided into five equal groups (A–E). The groups were A: (a harvested graft was adhered to 4-mm diameter defects), B: (same as group A with 6-mm diameter defect), C: (4- and 6-mm defects were made in either knee or the graft sutured), D: (only defects were created and remained intact), and E: (defects were created and filled with bioglue). The groups
were imaged 30 and 60 days postoperation. CT results showed that there was no significant difference in spur formation between groups, while focal osteopenia was higher in groupsA, B, C (30 days), and D (30 days). Sclerosis was lower in group E than other groups. MRI showed subchondral signal change and joint effusion to be lower in group E than other groups. The invented bioglue in the present work reduced OA signs on CT and MRI and had no side effects.
Objective: To study the influence of platelets on cartilage growth in articular defects in the sheep knee. Material and methods: Male Rasa Aragonesa sheep (6 months) were operated under general anaesthesia. A 4. mm diameter and 3. mm deep defect was made in the femoral trochlea in both knees. The right knee defect was filled with platelet concentrate 5. min after being activated with ClCa in group A (n=6), and similarly activated platelets + collagen scaffold in group B (n=6). Platelets were obtained by centrifuging 10. ml arterial blood from the sheep prior to the surgical procedure. The left knee defect was not filled. The sheep were sacrificed 10 weeks after surgery. Macroscopic and microscopic studies were performed. Results: In group A, hyaline cartilage was observed in the right knee defect at the end of the experiment in four cases. None of the defects of the left knees showed hyaline cartilage growth. In group B, hyaline cartilage was not observed in any right knee defect. However, in group B, all sheep showed better chondral cellularity and regeneration and lower fibrosis in the defects treated with platelets than in non-treated ones. Conclusions: This technique for articular defect reconstruction with platelets has shown satisfactory results in our study. However, collagen scaffolds may decrease this positive effect.
Objective
To study the influence of platelets on cartilage growth in articular defects in the sheep knee.
Thirty New Zealand white rabbits underwent anterior cruciate ligament (ACL) reconstruction in their right knees; 15 animals underwent a double-bundle anatomic ACL reconstruction using the medial third of the patellar tendon and the semitendinosus tendon. Additionally, 15 animals underwent ACL reconstruction, using a single-bundle semitendinosus tendon autograft. The knees of both groups were evaluated with a device similar to the KT1000 arthrometer onto which a dial indicator was attached (Mitutoyo dial indicator 2050) in 30 degrees and 90 degrees of flexion, preoperatively, after ACL resection and 3 months postoperatively. Statistical analysis of the results revealed that for 90 degrees of knee flexion, the mean estimated anterior shift for the double-bundle technique was 1.92 mm lesser than that of the single-bundle technique (P = 0.006). For 30 degrees of knee flexion, the mean anterior shift was again lesser than that of the single-bundle technique by 0.66 mm, but this difference was not statistically significant. The described double-bundle ACL reconstruction technique resulted in a more stable knee as far as the anterior tibial shift was concerned as compared to a single-bundle ACL reconstruction. This animal model may be potentially useful in the future for the study of other parameters influencing the outcome of the double-bundle ACL reconstruction.
Allografts of intact cartilage, isolated chondrocytes and cultured chondrocytes taken from the epiphysial growth-plate and from the articular surface of immature rabbits were inserted into full thickness defects in the tibial articular surface of 160 mature rabbits. In the contralateral knees, which were used as controls, similar defects were made but no grafts were inserted. Grafts were followed up for periods of up to one year after transplantation. Both intact articular and intact growth-plate grafts produced significantly better repair than that seen in control ungrafted defects in normal joints (P less than 0.01 and P less than 0.05 respectively) and in arthritic joints (P less than 0.01). Cultured chondrocytes cut to a precise fit also produced significantly better repair than ungrafted defects in arthritic joints (P less than 0.05).
Full-thickness defects of articular cartilage in the knee have a poor capacity for repair. They may progress to osteoarthritis and require total knee replacement. We performed autologous chondrocyte transplantation in 23 people with deep cartilage defects in the knee.
The patients ranged in age from 14 to 48 years and had full-thickness cartilage defects that ranged in size from 1.6 to 6.5 cm2. Healthy chondrocytes obtained from an uninvolved area of the injured knee during arthroscopy were isolated and cultured in the laboratory for 14 to 21 days. The cultured chondrocytes were then injected into the area of the defect. The defect was covered with a sutured periosteal flap taken from the proximal medial tibia. Evaluation included clinical examination according to explicit criteria and arthroscopic examination with a biopsy of the transplantation site.
Patients were followed for 16 to 66 months (mean, 39). Initially, the transplants eliminated knee locking and reduced pain and swelling in all patients. After three months, arthroscopy showed that the transplants were level with the surrounding tissue and spongy when probed, with visible borders. A second arthroscopic examination showed that in many instances the transplants had the same macroscopic appearance as they had earlier but were firmer when probed and similar in appearance to the surrounding cartilage. Two years after transplantation, 14 of the 16 patients with femoral condylar transplants had good-to-excellent results. Two patients required a second operation because of severe central wear in the transplants, with locking and pain. A mean of 36 months after transplantation, the results were excellent or good in two of the seven patients with patellar transplants, fair in three, and poor in two; two patients required a second operation because of severe chondromalacia. Biopsies showed that 11 of the 15 femoral transplants and 1 of the 7 patellar transplants had the appearance of hyaline cartilage.
Cultured autologous chondrocytes can be used to repair deep cartilage defects in the femorotibial articular surface of the knee joint.
Articular cartilage injuries and degeneration present a challenge for orthopedic surgeons. Chondrocytes have limited regenerative and reparative abilities. Healing of a defect results in a fibrocartilaginous repair tissue that lacks the structural and biomechanical properties of hyaline cartilage and that degrades over time. Polypeptide growth factors have an important role in regulating the behavior of all cells, including articular chondrocytes. Our understanding of growth factor effects on and interactions with chondrocytes is progressing rapidly. The most prominent growth factors identified for articular cartilage include insulin-like growth factor, fibroblast growth factor, the transforming growth factor-beta superfamily, hepatocyte growth factor, platelet-derived growth factor, Indian hedgehog and parathyroid hormone-related peptide, bone morphogenetic proteins, and the interleukin-1 receptor antagonist. Orthopedic surgeons need to be familiar with the properties of these growth factors, as they hold great therapeutic promise. In-progress clinical studies are examining how growth factors may have applications in treatments of bone.
The increase of the cytoplasmic Ca-concentration plays a central role in the initiation of platelet activation. Four kinds of movements of Ca-ions are presumed to occur during this process: a) Ca-ions liberated from membranes induce the rapid shape change, b) Vesicular organelles release Ca-ions into the cytoplasm which initiate the release reaction, c) The storage organelles called dense bodies, secrete their contents including Ca-ions to the outside during the release reaction, d) At the same time a rearrangement of the plasma membrane occurs, resulting in an increase in its permeability for Ca-ions as well as in an increase in the number of Ca-binding sites.
Since most processes occurring during platelet activation are reversible, the platelet must be equipped with a mechanism which removes Ca-ions from the cytoplasm. A vesicular fraction obtained from homogenized platelets indeed accumulates Ca actively. This Ca- pump is stimulated by cyclic AMP and protein kinase; it may be involved in the recovery of platelets after activation.
It becomes increasingly clear that the various manifestations of platelet activation are triggered by a rise in the cytoplasmic Ca2+-concentration. The evidence for this and possible mechanisms involved are discussed in some detail in the contributions by Detwiler et al. and by Gerrard and White to this symposium. In this article we shall discuss four different types of mobilization of Ca-ions which occur in the course of the activation of platelets. In addition, at least one transport step involved in the removal of Ca2+ must occur during relaxation of activated platelets.
Articular cartilage has a limited capacity for repair. We investigated the effect of rhBMP-2 (recombinant human bone morphogenetic protein-2) on the healing of full-thickness osteochondral defects in adult New Zealand White rabbits. A single defect, three millimeters wide by three millimeters deep, was created in the trochlear groove of the right femur in eighty-nine rabbits. The defect was either left empty, filled with a plain collagen sponge, or filled with a collagen sponge impregnated with five micrograms of rhBMP-2. The animals were killed at four, eight, or twenty-four weeks, and the repair tissue was examined histologically and evaluated with use of a grading scale. The defects also were examined immunohistochemically for the presence of type-II collagen at four and eight weeks. The rate of bone repair was evaluated with fluorescent labeling of bone at two and four weeks and with use of fluorescence microscopy at eight weeks. Treatment with rhBMP-2 greatly accelerated the formation of new subchondral bone and improved the histological appearance of the overlying articular surface. At twenty-four weeks, the thickness of the repair cartilage was 70 per cent that of the normal adjacent cartilage and a new tidemark usually had formed between the repair cartilage and the underlying subchondral bone. The average total scores on the histological grading scale were significantly better (p < 0.01) for the defects treated with rhBMP-2 than for the untreated defects (those left empty or filled with a plain collagen sponge) at all time-points. Immunostaining with an antibody against type-II collagen showed the diffuse presence of this cartilage-specific collagen throughout the repair cartilage in the treated defects. The untreated defects demonstrated minimum staining with this antibody.
Articular cartilage has a limited capacity for repair. In recent clinical and animal experiments, investigators have attempted to elicit the repair of defects of articular cartilage by injecting cultured autologous chondrocytes under a periosteal flap (a layer of periosteum). The objective of the present study was to determine the effect of cultured autologous chondrocytes on healing in an adult canine model with use of histomorphometric methods to assess the degree of repair. A total of forty-four four-millimeter-diameter circular defects were created down to the zone of calcified cartilage in the articular cartilage of the trochlear groove of the distal part of the femur in fourteen dogs. The morphology and characteristics of the original defects were defined in an additional six freshly created defects in three other dogs. Some residual noncalcified articular cartilage, occupying approximately 2 per cent of the total cross-sectional area of the defect, was sometimes left in the defect. The procedure sometimes damaged the calcified cartilage, resulting in occasional microfractures or larger fractures, thinning of the zone of calcified cartilage, or, rarely, small localized penetrations into subchondral bone. The forty-four defects were divided into three treatment groups. In one group, cultured autologous chondrocytes were implanted under a periosteal flap. In the second group, the defect was covered with a periosteal flap but no autologous chondrocytes were implanted. In the third group (the control group), the defects were left empty. The defects were analyzed after twelve or eighteen months of healing. Histomorphometric measurements were made of the percentage of the total area of the defect that became filled with repair tissue, the types of tissue that filled the defect, and the integration of the repair tissue with the adjacent cartilage at the sides of the defects and with the calcified cartilage at the base of the defect. In histological sections made through the center of the defects in the three groups, the area of the defect that filled with new repair tissue ranged from a mean total value of 36 to 76 per cent, with 10 to 23 per cent of the total area consisting of hyaline cartilage. Integration of the repair tissue with the adjacent cartilage at the edges of the defect ranged from 16 to 32 per cent in the three groups. Bonding between the repair tissue and the calcified cartilage at the base of the defect ranged from 41 to 89 per cent. With the numbers available, we could detect no significant difference among the three groups with regard to any of the parameters used to assess the quality of the repair. In the two groups in which a periosteal flap was sutured to the articular cartilage surrounding the defect, the articular cartilage showed degenerative changes that appeared to be related to that suturing.
Optimal repair of chondral defects is likely to require both a suitable population of chondrogenic cells and a biodegradable matrix to provide a space-filling structural support during the early stages of cartilage formation. This study examined the ability of chondrocytes to support cartilage formation when incorporated into biodegradable scaffolds constructed from copolymers (PLG) of polylactic acid (PLA) and polyglycolic acid (PGA) and implanted in the calf muscle of nude mice. Scaffolds were fabricated to be more hydrophilic (PLG-H) or were reinforced with 10% PGA fibers (PLG-FR), increasing the stiffness of the implant by 20-fold. Confluent primary cultures of rat costochondral resting zone chondrocytes (RC) were loaded into PLG-H foams and implanted intramuscularly. To determine if growth factor pretreatment could modulate the ability of the cells to form new cartilage, RC cells were pretreated with recombinant human platelet derived growth factor-BB IPDGF-BB) for 4 or 24 h prior to implantation. To assess whether scaffold material properties could affect the ability of chondrogenic cells to form cartilage, RC cells were also loaded into PLG-FR scaffolds. To determine if the scaffolds or treatment with PDGF-BB affected the rate of chondrogenesis, tissue at the implant site was harvested at four and eight weeks post-operatively, fixed, decalcified and embedded in paraffin. Sections were obtained along the transverse plane of the lower leg, stained with haematoxylin and eosin, and then assessed by morphometric analysis for area of cartilage, area of residual implant, and area of fibrous connective tissue formation (fibrosis). Whether or not the cartilage contained hypertrophic cells was also assessed. The amount of residual implant did not change with time in any of the implanted tissues. The area occupied by PLG-FR implants was greater than that occupied by PLG-H implants at both time points. All implants were surrounded by fibrous connective tissue, whether they were seeded with RC cells or not. The amount of fibrosis was reduced at eight weeks for both implant types. When RC cells were present, the amount of fibrosis was less than seen in cell-free scaffolds. Pretreatment with PDGF-BB caused a slightly greater degree of fibrosis at four weeks than was seen if untreated cells were used in the implants. However, at eight weeks, if the cells had been exposed to PDGF-BB for 24 h, fibrosis was comparable to that seen associated with cell-free scaffolds. The cells supported an equivalent area of cartilage formation in both scaffolds. PDGF-BB caused a time-dependent decrease in cartilage formation at four weeks, but at eight weeks, there was a marked increase in cartilage formation in PDGF-BB-treated cells that was greatest in cells exposed for 4 h compared to those exposed for 24 h. Moreover, PDGF-BB decreased the formation of hypertrophic cells. The results indicate that in this model, RC cells produce cartilage; pretreatment of the RC cells with PDGF-BB promotes retention of a hyaline-like chondrogenic phenotype; and the material properties of the implant do not negatively impact on the ability of the cells to support chondrogenesis.
Recently proposed procedures for in vitro generation of new cartilage may be difficult to perform in humans because so many chondrocytes are needed for tissue engineering. In this study the authors investigated new, efficient, low-cost techniques for the isolation and culture of chondrocytes from the ear cartilage of the rabbit. They performed a low-density monolayer culture with a low concentration (0.5%, 1%) of human platelet supernatant and observed cell proliferation (seeding efficiency, deoxyribonucleic acid synthesis), matrix synthesis (glycosaminoglycan synthesis), and the expression of type I and type II collagen (reverse transcriptase polymerase chain reaction). Seeding efficiency was increased in 1% of platelet supernatant-treated cultures by two to three times compared with untreated controls. One percent platelet supernatant had increased the incorporation of [3H]-thymidine by 1.9 to 2.5 times at 72 hours compared with controls. Glycosaminoglycan synthesis was increased in platelet supernatant-treated chondrocytes at 96 hours compared with controls. Chondrocytes treated with 1% platelet supernatant showed a decreased expression of the type II collagen gene. Supplementation with a high concentration (10%) of the platelet supernatant provided the conditions for in vitro chondrocyte mass formation. These results indicate that proliferation and matrix synthesis of auricular chondrocytes is stimulated by a low concentration of platelet supernatant. On the other hand, chondrocytes were immobilized by a high concentration of platelet supernatant. Platelet supernatant may be useful as an inexpensive autologous source of multiple growth factors to enhance chondrocyte proliferation, and also may play the role of scaffold for chondrocytes. Additional investigation is underway to generate culture conditions that promote the differentiation as well as the proliferation of chondrocytes.
Variations in certain mesenchymal tissue healing processes are not widely recognized. The current review summarizes key differences in healing mechanisms and healing potential after injury to soft tissues having different healing outcomes.
The aim of this study was to investigate the effects of different growth factors on the chondrogenic potential of human bone marrow stromal cells (BMSC).
Different growth factors which have been shown to sustain the osteogenic potential of BMSC during their 'in vitro' expansion were assayed for the maintenance of the chondrogenic potential. We compared the ability of BMSC to reconstitute cartilage in vitro with their ability to form bone on hydroxyapatite microporous particles in an ectopic bone formation assay.
Among the factors assayed, fibroblast growth factor 2 (FGF2) was the most effective in promoting growth of BMSC 'in vitro'. For all growth factors tested, we have found a complete overlap of the enhancement of chondrogenic and osteogenic potential. Any factor, either promoting or depressing bone formation, exerted the same effect on the chondrogenic potential of human BMSC. In particular, FGF2, either alone or in combination with other factors, strongly supported the formation of bone as well as of cartilage.
We conclude that FGF2 maintains human BMSC in an immature state allowing their 'in vitro' expansion. Expanded cells retain the chondro- osteogenic potential. Interestingly, the chondrogenic potential of BMSC 'in vitro' is directly related to their ability to form bone 'in vivo'. BMSC expanded 'ex vivo' are presently being proposed for cell therapy of bone defects. 'In vitro' chondrogenesis may be regarded as a rapid prediction assay to assess cell ability to form bone after 'in vivo' transplant.
The objective of the study was to evaluate the growth-promoting activity of human platelet supernatant on primary chondrocytes in comparison with fetal calf serum (FCS) supplemented cell culture medium. Furthermore, the differentiation potential of platelet supernatant was determined in three-dimensional artificial cartilage tissues of bovine articular chondrocytes. Proliferation of articular and nasal septal chondrocytes was assayed by incorporation of BrdU upon stimulation with ten different batches of human platelet supernatant. On bovine articular chondrocytes, all these batches were at least as growth-promoting as FCS. On nasal septal chondrocytes, nine out of ten batches revealed increased or equivalent mitogenic stimulation compared with medium supplemented with FCS. Three-dimensional culture and subsequent histological analysis of matrix formation were used to determine the differentiation properties of platelet supernatant on articular chondrocytes. Human platelet supernatant failed to induce the deposition of typical cartilage matrix components, whereas differentiation and matrix formation were apparent upon cultivation of articular chondrocytes with FCS. Proliferation assays demonstrated that human platelet supernatant stimulates growth of articular and nasal septal chondrocytes; however, platelet supernatant failed to stimulate articular chondrocytes to redifferentiate in three-dimensional chondrocyte cultures. Therefore platelet lysate may be suitable for chondrocyte expansion, but not for maturation of tissue-engineered cartilage.
We have performed a prospective, single-surgeon study analysing the histological results of autologous chondrocyte implantation.
Fourteen patients underwent autologous chondrocyte implantation of the knee and were evaluated at one year by clinical assessment and arthroscopy. Standard staining was used to examine the sections. In addition, in situ hybridisation was used to establish type-IIa and type-IIb collagen mRNA expression and immunolocalisation techniques demonstrated the positions of type-II and type-X collagen.
Eight patients regenerated hyaline cartilage and also contained type-X collagen in the deepest layers and type-II collagen in the deep layers. Three demonstrated fibrocartilage and had type-II collagen in the deep layers. In situ hybridisation revealed that all 14 samples had the potential to express both type-IIa and type-IIb collagen.
We have shown that one year after the initial implantation chondrocytes are capable of producing type-II collagen and that they continue to proliferate and mature.
Circulating tissue factor accumulates in the developing thrombus and contributes to fibrin clot formation. To determine whether tissue factor derived from hematopoietic cells is delivered to the thrombus via tissue factor-bearing microparticles or circulating leukocytes expressing tissue factor on the plasma membrane, we compared the kinetics of tissue factor accumulation in the developing arteriolar thrombus with the time course of leukocyte-thrombus interaction and microparticle-thrombus interaction in the microcirculation of a living mouse using intravital high-speed widefield and confocal microscopy. Tissue factor rapidly accumulated in the developing thrombus, appearing immediately following vessel wall injury, reaching a first peak in approximately 100 s. In contrast, leukocyte-thrombus interaction was not observed until after 2-3 min following vessel wall injury. Maximal leukocyte rolling and firm leukocyte adherence on thrombi in wild-type mice were observed after approximately 8 min and were dependent on P-selectin and P-selectin glycoprotein ligand-1. This delay in P-selectin-dependent leukocyte rolling is a result of time-dependent platelet activation and P-selectin expression on the luminal surface of the thrombus. In contrast, microparticle accumulation in the developing arteriolar thrombus was rapid, and peak accumulation was within 60 s. The accumulation of hematopoietic cell-derived tissue factor in the developing thrombus correlates to the kinetics of microparticle accumulation and does not correlate temporally with leukocyte-thrombus interaction. These results indicate that tissue factor derived from hematopoietic cells is delivered by microparticles during the initial phase of thrombus development in vivo.
To examine thrombus formation in a living mouse, new technologies involving intravital videomicroscopy have been applied to the analysis of vascular windows to directly visualize arterioles and venules. After vessel wall injury in the microcirculation, thrombus development can be imaged in real time. These systems have been used to explore the role of platelets, blood coagulation proteins, endothelium, and the vessel wall during thrombus formation. The study of biochemistry and cell biology in a living animal offers new understanding of physiology and pathology in complex biologic systems.
Specimen obtained 6 weeks after surgery from platelet-treated cartilaginous defect, 2 min after platelet activation (right knee). Hematoxylin-eosin
- Fig
Fig. 5. Specimen obtained 6 weeks after surgery from platelet-treated cartilaginous defect, 2 min after platelet activation (right knee). Hematoxylin-eosin, ×10.
A method of resurfacing osteoarthritic knee joints
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Leucocyte versus microparticle-mediated tissue factor transfer during arteriolar thrombus development.
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