Pretreatment of periosteum with TGF-beta1 in situ enhances the quality of osteochondral tissue regenerated from transplanted periosteal grafts in adult rabbits.

OBJECTIVE: To compare the efficacy of in situ transforming growth factor-beta1 (TGF-beta1)-pretreated periosteum to untreated periosteum for regeneration of osteochondral tissue in rabbits. METHODS: In the pretreatment group, 12 month-old New Zealand white rabbits received subperiosteal injections of 200 ng of TGF-beta1 percutaneously in the medial side of the proximal tibia, 7 days prior to surgery. Control rabbits received no treatment prior surgery. Osteochondral transverse defects measuring 5mm proximal to distal and spanning the entire width of the patellar groove were created and repaired with untreated or TGF-beta1-pretreated periosteal grafts. Post-operatively the rabbits resumed normal cage activity for 6 weeks. RESULTS: Complete filling of the defects with regenerated tissue was observed in both the TGF-beta1-pretreated and control groups with reformation of the original contours of the patellar groove. The total histological score (modified O'Driscoll) in the TGF-beta1-pretreated group, 20 (95% Confidence Interval (CI), 19-21), was significantly higher (P=0.0001) than the control group, 18 (16-19). The most notable improvements were in structural integrity and subchondral bone regeneration. No significant differences in glycosaminoglycan or type II collagen content, or equilibrium modulus were found between the surgical groups. The cambium of the periosteum regenerated at the graft harvest site was significantly thicker (P=0.0065) in the TGF-beta1-pretreated rabbits, 121 microm (94-149), compared to controls, 74 microm (52-96), after 6 weeks. CONCLUSIONS: This study demonstrates that in situ pretreatment of periosteum with TGF-beta1 improves osteochondral tissue regeneration at 6-weeks post-op compared to untreated periosteum in 12 month-old rabbits.

Tissue engineering of cartilage using poly-epsilon-caprolactone nanofiber scaffolds seeded in vivo with periosteal cells.

OBJECTIVE: To determine the potential of periosteal cells to infiltrate poly-epsilon-caprolactone (PCL) nanofiber scaffolds in vivo and subsequently produce cartilage in vitro. DESIGN: PCL nanofiber scaffolds, with or without chitosan-coating were implanted under periosteum in 6-month-old rabbits. Transforming growth factor-beta1 (TGF-beta1) or vehicle was injected into each implant site. After 1, 3, 5 or 7 days, scaffolds were removed, separated from the periosteum, and the scaffolds and periosteum were cultured separately for 6 weeks under chondrogenic conditions. Sulfated glycosaminoglycan (GAG), type II collagen, DNA content, cartilage yield, and calcium deposition were then analyzed. RESULTS: Cell infiltration was observed in all scaffolds. Cartilage formation in the uncoated scaffolds increased with duration of implantation (maximum at 7 days). Cells in the uncoated scaffolds implanted for 7 days produced significantly higher levels of both GAG [560 (95% confidence interval (CI), 107-1013) vs 228 (95% CI, 177-278) microg GAG/microg DNA] and cartilage yield [9% (95% CI, 3-14%) vs 0.02% (95% CI, 0-0.22%)] compared to chitosan-coated scaffolds (P=0.006 or less). There was no significant difference in GAG content or cartilage yield between the TGF-beta1-injected and vehicle-injected scaffolds. However, significantly more mineral deposition was detected in TGF-beta1-injected scaffolds compared to vehicle-injected scaffolds (P<0.0001). Cartilage yield from the periosteum, moreover, was significantly increased by subperiosteal TGF-beta1 injections (P<0.001). However, this response was reduced when chitosan-coated scaffolds were implanted. CONCLUSIONS: This study demonstrates that it is possible to seed PCL nanofiber scaffolds with periosteal cells in vivo and subsequently produce engineered cartilage in vitro.

Rejuvenation of periosteal chondrogenesis using local growth factor injection.

OBJECTIVE: To examine the potential for rejuvenation of aged periosteum by local injection of transforming growth factor-beta1 (TGF-beta1) and insulin-like growth factor-1 (IGF-1) alone or in combination to induce cambium cell proliferation and enhance in vitro periosteal cartilage formation. METHODS: A total of 367 New Zealand white rabbits (6, 12, and 24+ month-old) received subperiosteal injections of TGF-beta1 and/or IGF-1 percutaneously. After 1, 3, 5, or 7 days, the rabbits were sacrificed and cambium cellularity or in vitro cartilage forming capacity was determined. RESULTS: A significant increase in cambium cellularity and thickness, and in vitro cartilage formation was observed after injection of TGF-beta1 alone or in combination with IGF-1. In 12 month-old rabbits, mean cambium cellularity increased 5-fold from 49 to 237 cells/mm and in vitro cartilage production increased 12-fold from 0.8 to 9.7 mg 7 days after TGF-beta1 (200 ng) injection compared to vehicle controls (P<0.0001). A correlation was observed between cambium cellularity and in vitro cartilage production (R2=0.98). An added benefit of IGF-1 plus TGF-beta1 on in vitro cartilage production compared to TGF-beta1 alone was observed in the 2 year-old rabbits. IGF-1 alone generally had no effect on either cambium cellularity or in vitro cartilage production in any of the age groups. CONCLUSIONS: These results clearly demonstrate that it is possible to increase cambium cellularity and in vitro cartilage production in aged rabbit periosteum, to levels comparable to younger rabbits, using local injection of TGF-beta1 alone or in combination with IGF-1, thereby rejuvenating aged periosteum.

The role of the posterior bundle of the medial collateral ligament in posteromedial rotatory instability of the elbow.

Aims: The aim of this study was to evaluate two hypotheses. First, that disruption of posterior bundle of the medial collateral ligament (PMCL) has to occur for the elbow to subluxate in cases of posteromedial rotatory instability (PMRI) and second, that ulnohumeral contact pressures increase after disruption of the PMCL. Materials and Methods: Six human cadaveric elbows were prepared on a custom-designed apparatus which allowed muscle loading and passive elbow motion under gravitational varus. Joint contact pressures were measured sequentially in the intact elbow (INTACT), followed by an anteromedial subtype two coronoid fracture (COR), a lateral collateral ligament (LCL) tear (COR + LCL), and a PMCL tear (COR + LCL + PMCL). Results: There was no subluxation or joint incongruity in the INTACT, COR, and COR + LCL specimens. All specimens in the COR + LCL + PMCL group subluxated under gravity-varus loads. The mean articular contact pressure of the COR + LCL group was significantly higher than those in the INTACT and the COR groups. The mean articular contact pressure of the COR + LCL + PMCL group was significantly higher than that of the INTACT group, but not higher than that of the COR + LCL group. Conclusion: In the presence of an anteromedial fracture and disruption of the LCL, the posterior bundle of the MCL has to be disrupted for gross subluxation of the elbow to occur. However, elevated joint contact pressures are seen after an anteromedial fracture and LCL disruption even in the absence of such subluxation. Cite this article: Bone Joint J 2018;100-B:1060-5.

Porous tantalum biocomposites for osteochondral defect repair: A follow-up study in a sheep model.

OBJECTIVES: We sought to determine if a durable bilayer implant composed of trabecular metal with autologous periosteum on top would be suitable to reconstitute large osteochondral defects. This design would allow for secure implant fixation, subsequent integration and remodeling. MATERIALS AND METHODS: Adult sheep were randomly assigned to one of three groups (n = 8/group): 1. trabecular metal/periosteal graft (TMPG), 2. trabecular metal (TM), 3. empty defect (ED). Cartilage and bone healing were assessed macroscopically, biochemically (type II collagen, sulfated glycosaminoglycan (sGAG) and double-stranded DNA (dsDNA) content) and histologically. RESULTS: At 16 weeks post-operatively, histological scores amongst treatment groups were not statistically different (TMPG: overall 12.7, cartilage 8.6, bone 4.1; TM: overall 14.2, cartilage 9.5, bone 4.9; ED: overall 13.6, cartilage 9.1, bone 4.5). Metal scaffolds were incorporated into the surrounding bone, both in TM and TMPG. The sGAG yield was lower in the neo-cartilage regions compared with the articular cartilage (AC) controls (TMPG 20.8/AC 39.5, TM 25.6/AC 33.3, ED 32.2/AC 40.2 µg sGAG/1 mg respectively), with statistical significance being achieved for the TMPG group (p < 0.05). Hypercellularity of the neo-cartilage was found in TM and ED, as the dsDNA content was significantly higher (p < 0.05) compared with contralateral AC controls (TM 126.7/AC 71.1, ED 99.3/AC 62.8 ng dsDNA/1 mg). The highest type II collagen content was found in neo-cartilage after TM compared with TMPG and ED (TM 60%/TMPG 40%/ED 39%). Inter-treatment differences were not significant. CONCLUSIONS: TM is a highly suitable material for the reconstitution of osseous defects. TM enables excellent bony ingrowth and fast integration. However, combined with autologous periosteum, such a biocomposite failed to promote satisfactory neo-cartilage formation.Cite this article: E. H. Mrosek, H-W. Chung, J. S. Fitzsimmons, S. W. O'Driscoll, G. G. Reinholz, J. C. Schagemann. Porous tantalum biocomposites for osteochondral defect repair: A follow-up study in a sheep model. Bone Joint J 2016;5:403-411. DOI: 10.1302/2046-3758.59.BJR-2016-0070.R1.

Familial recurrence of achondroplasia.

A family is reported among whom 2 second-cousins-once removed (two half or half third cousins) demonstrate clinical and radiological features of classical achondroplasia.

Method for automated cartilage histomorphometry.

We have developed and tested a color-based method for automated computerized histomorphometric analysis of cartilage. Histological sections stained with safranin O from 29 rabbit periosteal agarose-cultured explants were selected with various amounts of cartilage (0-100%). Color photomicrographs of these sections were visually assessed by five expert observers who estimated the percent area occupied by cartilage and outlined (in pen) the areas they considered to be cartilage. Manual histomorphometry was performed by cutting out and weighing the outlined areas. The average area for each of the five observers ranged from 31% to 43% (intraclass correlation coefficient = 0.70). The average of these values was used as a "gold standard" against which to compare the computer measurements. When point counting histomorphometry was performed on the 29 sections, the data agreed with the measurements made by the other five cartilage experts (r2 = 0.96; p < 0.0001). The analysis of cartilage is based on safranin O stain, using a custom-designed Vidas 2.1 Image Analysis Program (Zeiss). The computer-based results correlated very closely with those obtained by manual (p = 0.0001; r2 = 0.92) and point counting (r2 = 0.92; p < 0.0001) histomorphometry. The mean percentage of the sections occupied by cartilage measured in the automated mode was only 6% higher than that using the gold standard. Histological complexity had only a minor effect on the computerized values. The automated computerized image analysis system has the advantages of objectivity, accuracy, repeatability, and ease of use.

Validation of a simple histological-histochemical cartilage scoring system.

In this study, we assessed the validity of a subjective histological-histochemical scoring system as compared to an automated histomorphometry program for analyzing cartilage repair tissue. In the first part of the study, we assessed the ability of the human eye to estimate the percent cartilage in a histological section. Twenty-nine rabbit periosteal explants that had been cultured in agarose transforming growth factor-beta (TGF-beta) were selected so that the percentage of cartilage in the specimens was distributed equally from 0% to 100%. Color photomicrographs were evaluated by 5 expert observers who gave a visual estimate of the percent cartilage. There was a strong correlation between the estimated and actual percent cartilage (R(2) = 0.92, p < 0.0001) and among the observers (I.C.C. = 0.89). On average, the estimated percent cartilage was within ten percent of the actual percent measured. In the second part, we compared the data derived using a simple cartilage score with those obtained by automated image analysis. The histological slides from 159 explants cultured under various experimental conditions (14 treatment groups) in two different experiments were analyzed. The cartilage content was estimated visually and a score from 0 to 3 was assigned. A previously validated, computerized image analysis system was used to measure the actual percent cartilage. Statistical analyses revealed a good linear regression (R(2) = 0.84, p = 0.0001), and even better polynomial correlation between the actual measurement and the score (R(2) = 0.88, p = 0.0001). These data demonstrate the validity of a simple histological-histochemical subjective scoring system. A computerized automated program such as the one employed in this study is preferable due to its many advantages. However, a subjective scoring system may be appropriate to use when the funding and expertise required for a computerized image analysis program are not available.

Viability of periosteal tissue obtained postmortem.

Periosteal autografts have the potential to regenerate articular cartilage defects, but this potential is limited by the patient's age. Allograft transplantation from a young donor to an older recipient might bypass this limitation. The effect of the time delay, between death and harvesting of a periosteal graft, on the chondrogenic potential of periosteum is important not only for transplantation but also for studies dealing with tissues retrieved postmortem (i.e., including the periosteal explant model). The purpose of this study was to investigate the chondrogenic potential of periosteum obtained postmortem and a possible beneficial effect of hypothermia. Thirty NZ white rabbits (2 months old) were sacrificed and stored at room temperature or 4 degrees C for 0, 4, 6, 8, 12, 16, 18, or 24 h. Periosteal explants were then obtained and a standard cartilage yield assay performed by culturing them for 6 weeks using the periosteal organ culture model as previous published. TGF-beta1 (10 ng/ml) was added for the first 14 days of culture. Histochemical analysis and quantitative collagen typing were performed. In the explants from the animals kept for 4 h at room temperature growth and chondrogenesis were dramatically reduced. Little or no chondrogenesis was seen in explants from rabbits maintained at room temperature after 4-8 h (or more) postmortem. Cooling the rabbits to 4 degrees C partially prevented this loss of viability and continued to do so for 24 h. Even storage at 4 degrees C did not eliminate the decrease in chondrogenic potential, though it did permit partial preservation of chondrogenic potential. If periosteum is to be used for allograft transplantation, or if it is used for experimental study, its viability must be assured. This is best accomplished by harvesting it immediately postmortem. Preservation techniques, cryopreservation, or hypothermia might be useful in preserving periosteal chondrogenic potential.

Isolation of a cDNA sequence of rabbit GDF5 (mature form) and pattern of its mRNA expression during periosteal chondrogenesis.

Articular cartilage has a limited ability for repair and/or regeneration. Periosteal grafts, having chondrogenic potential, are used clinically and in experimental models to study the repair and regeneration of cartilage. Growth/differentiation factor 5 (GDF5), recently shown to be involved in chondrogenesis and normal skeletal development, is a bioactive candidate for augmenting the repair of damaged cartilage. In order to investigate the role of GDF5 during periosteal chondrogenesis, the rabbit sequence must be known, as most experimental models involve rabbit tissues. For this purpose, the complete rabbit-specific cDNA sequence of the mature form of GDF5 was determined. Mature rabbit GDF5 was found to be 100% identical to that of human GDF5 at the amino acid level. Using the cDNA sequence, specific primers for PCR were designed. Quantitative RT-PCR, using rabbit-specific primers, showed up-regulation of GDF5 mRNAs early during periosteal chondrogenesis suggesting its potential involvement in this process. The timing and magnitude of this expression was markedly stimulated by TGF-beta 1, which has already been shown to be a potent inducer of periosteal chondrogenesis.

Gene digging. A method for obtaining species-specific sequence based on conserved segments of nucleotides in open reading frames.

A method termed "gene digging" has been developed based on our observation of stretches of highly conserved nucleotide sequence in the coding region of many genes across related species. Rabbit-specific nucleotide sequences corresponding to desired coding segments of 14 different genes were obtained with primers that were designed based on conserved nucleotide stretches. Our success in gene digging could be attributable to the method's inherent ability to reduce the degeneracy of primers by more than two orders of magnitude (sometimes by more than three orders of magnitude) compared to primers designed from conserved amino acids. Our results not only demonstrate the value of the method, but also hint at a thus far unknown functional significance of conserved nucleotide stretches in the coding region of various genes. In our hands the method worked 14 out of 14 times indicating generality of the concept.

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.

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-beta 1 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.

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.

Histomorphological and proliferative characterization of developing periosteal neochondrocytes in vitro.

Periosteal chondrogenesis is relevant to cartilage repair and fracture healing. Cell proliferation is a limiting factor of cartilage production. We used an in vitro organ culture model to test the hypothesis that proliferative activity correlates with cell morphology. One hundred and four periosteal explants from 26 two-month old New Zealand rabbits were cultured for up to 42 days. They were analyzed histomorphologically, and immunohistochemically with proliferative cell nuclear antigen (PCNA). The periosteal neocartilage displayed a consistent zonal pattern of chondrocyte cell shapes. The flat cell zone from day 7 to 21, consisted of uniform-sized small spindle-shaped cells. The round cell zone, which appeared on day 14, consisted of variable-sized round cells averaging 510 +/- 250 microm2 in area. They were subdivided into small round (<510 microm2) and large round cells (>510 microm2). The proliferative index was highest in the small round cell group (32 +/- 6%), intermediate in the flat cell group (27 +/- 6%), and lowest in the large round cell group (20 +/- 7%) (P < 0.001). Furthermore, the proliferative indices in the round cell group were inversely proportional to cell size. Therefore, (1) there is a sequential progression of cell morphology during periosteal chondrogenesis, (2) cell differentiation is arrested prior to terminal differentiation for some cells and not for others, and (3) proliferative activity is strongly related to cell morphology. This organ culture model provides us with opportunities to study the regulation of terminal chondrocyte differentiation and the control of cell proliferation. This will contribute to our understanding of cartilage repair, fracture healing and growth plate physiology.

Initial evidence for the involvement of bone morphogenetic protein-2 early during periosteal chondrogenesis.

The potential of periosteum to form cartilage makes periosteal transplantation a viable approach to repairing defects in articular cartilage, which has a limited potential for repair. However, cartilage repair, including that by periosteal chondrogenesis, is poorly understood. Consequently, a thorough understanding of its molecular mechanisms will help to achieve the quality of neocartilage required for its clinical application in damaged joints. An in vitro model was used to study the early molecular events of periosteal chondrogenesis. During the search for the expression of transforming growth factor-beta-related mRNAs in this model system, bone morphogenetic protein-2 mRNA expression was found to be upregulated 20-fold within the first 12 hours of culture. This stimulation was dependent on the explants being suspended in agarose and did not occur with explants cultured in liquid medium. The upregulation of bone morphogenetic protein-2 mRNA expression was also enhanced by exogenously added transforming growth factor-beta1 in the presence of fetal calf serum. The upregulation, however, was not transient; rather, it persisted over a prolonged period in both transforming growth factor-beta1-treated and untreated explants. Further data indicate that this stimulation of bone morphogenetic protein-2 mRNA expression was regulated at the transcriptional level and that no new protein synthesis was required for this. Bone morphogenetic protein-2 is known to influence developmental chondrogenesis; therefore, these observations direct our attention toward an important potential role of it as a regulator of the early events in cartilage repair. Furthermore, because periosteum produces fracture (cartilage) callus, these findings may be important in defining the molecular mechanisms of fracture healing.

Poly-epsilon-caprolactone/gel hybrid scaffolds for cartilage tissue engineering.

The aim of this study was to determine the suitability of hybrid scaffolds composed of naturally derived biopolymer gels and macroporous poly-epsilon-caprolactone (PCL) scaffolds for neocartilage formation in vitro. Rabbit articular chondrocytes were seeded into PCL/HA (1 wt % hyaluronan), PCL/CS (0.5 wt % chitosan), PCL/F (1:3 fibrin sealant plus aprotinin), and PCL/COL1 (0.24% type I collagen) hybrids and cultured statically for up to 50 days. Growth characteristics were evaluated by histological analysis, scanning electron microscopy, and confocal laser scanning microscopy. Neocartilage was quantified using a dimethyl-methylene blue assay for sulfated glycosaminoglycans (sGAG) and an enzyme-linked immunosorbent assay for type II collagen (COL2), normalized to dsDNA content by fluorescent PicoGreen assay. Chondrocytes were homogenously distributed throughout the entire scaffold and exhibited a predominantly spheroidal shape 1 h after being seeded into scaffolds. Immunofluorescence depicted expanding proteoglycan deposition with time. The sGAG per dsDNA increased in all hybrids between days 25 and 50. PCL/HA scaffolds consistently promoted highest yields. In contrast, total sGAG and total COL2 decreased in all hybrids except PCL/CS, which favored increasing values and a significantly higher total COL2 at day 50. Overall, dsDNA content decreased significantly with time, and particularly between days 3 and 6. The PCL/HA hybrid displayed two proliferation peaks at days 3 and 25, and PCL/COL1 displayed one proliferation peak at day 12. The developed hybrids provided distinct short-term environments for implanted chondrocytes, with not all of them being explicitly beneficial (PCL/F, PCL/COL1). The PCL/HA and PCL/CS hybrids, however, promoted specific neocartilage formation and initial cell retention and are thus promising for cartilage tissue engineering.

Classroom teaching in genetics and birth defects: the Nottingham experience.

Advances in genetics and their implications for disease prevention make it important that there should be more emphasis on the teaching of human genetics in our schools. This is likely to require cooperation between educationalists and interested medical staff and two such ventures, one in North America and the other in Nottinghamshire, are described. The latter, involving school teachers, the Department of Health Education, and the staff of the Clinical Genetic Service appears to meet a need, and the teaching material developed is in use in secondary schools throughout the county. The teaching package contains simple examples of disease inheritance, brief information about antenatal diagnosis and genetic counselling, and some class exercises designed to encourage discussion on handicap and its effects on the family.