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.

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.

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.

Tuberculosis stigma as a social determinant of health: a systematic mapping review of research in low incidence countries.

Tuberculosis (TB)-related stigma is an important social determinant of health. Research generally highlights how stigma can have a considerable impact on individuals and communities, including delays in seeking health care and adherence to treatment. There is scant research into the assessment of TB-related stigma in low incidence countries. This study aimed to systematically map out the research into stigma. A particular emphasis was placed on the methods employed to measure stigma, the conceptual frameworks used to understand stigma, and whether structural factors were theorized. Twenty-two studies were identified; the majority adopted a qualitative approach and aimed to assess knowledge, attitudes, and beliefs about TB. Few studies included stigma as a substantive topic. Only one study aimed to reduce stigma. A number of studies suggested that TB control measures and representations of migrants in the media reporting of TB were implicated in the production of stigma. The paucity of conceptual models and theories about how the social and structural determinants intersect with stigma was apparent. Future interventions to reduce stigma, and measurements of effectiveness, would benefit from a stronger theoretical underpinning in relation to TB stigma and the intersection between the social and structural determinants of health.

The effect of the orientation of the radial head on the kinematics of the ulnohumeral joint and force transmission through the radiocapitellar joint.

Background. The treatment of radial head fractures that are not amenable to an open reduction and internal fixation, remains to be a difficult issue. A potential problem with prosthetic replacement of the radial head is the shape of current radial head prostheses. The purpose of this study was to determine the effect of the shape of the radial head on kinematics and load transfer of the elbow. Methods. Kinematics of the elbow and radiocapitellar force transmission were measured in 6 fresh frozen upper extremities. The effect of radial head shape was tested by rotating the head 90 degrees , with a custom-made 'native' radial head prosthesis. 3-D spatial orientation of the ulna showed an average difference in ulnohumeral laxity, between the nominal and 90 degrees conditions, of 0.1 degrees throughout the arc of motion with neutral forearm rotation (maximum: 2 degrees ). Findings. We found an average difference in ulnar axial rotation, of 0.1 degrees (maximum: 1.9 degrees ). No differences showed statistical significance. Radiohumeral joint force was measured and maximally showed a 32 times increase of force in the altered shape conditions. Interpretation. Our results show that the kinematics of the elbow was not affected by altering the shape of the radial head, but it did adversely affect the forces in the radiohumeral joint. This could possibly generate degenerative changes in the elbow.

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.

Intrinsic constraint of unlinked total elbow replacements--the ulnotrochlear joint.

BACKGROUND: Many unlinked total elbow replacement designs with radically differing articular geometries exist, suggesting that there is no consensus regarding an optimal design. A feature inherent to the articular design is the intrinsic constraint afforded to the joint by the implant. Our aim was to compare the intrinsic constraints of unlinked implants with that of the normal ulnotrochlear joint. METHODS: We tested twelve cadaveric ulnotrochlear joints with a custom-made multiple-axis materials testing machine. With compressive loads ranging from 10 to 100 N, the joints were moved in either valgus or varus directions at 90 degrees of flexion. The ulnotrochlear components from a single example of five medium-sized unlinked elbow replacements (Ewald, Kudo, Pritchard ERS, Sorbie-Questor, and Souter-Strathclyde) were also tested. The recorded measurements included the torques and forces, angular displacement, and axial displacement of the humerus relative to the ulna. RESULTS: In general, the peak torque and the constraint ratio significantly increased with increasing compressive load for the implants as well as for the normal elbow. In valgus displacement, the Souter-Strathclyde implant had the highest and the Sorbie-Questor had the smallest peak torque and the Souter-Strathclyde had the highest and the Ewald had the smallest constraint ratio. In varus displacement, the Kudo had the highest and the Ewald had the smallest peak torque and constraint ratio. CONCLUSIONS: The constraint ratio is a characteristic that is useful for describing elbow joint behavior and for comparing the behavior of implants with that of the human elbow. Of the unlinked implants tested, the Souter-Strathclyde and Kudo prostheses most closely approximated the behavior of the human elbow joint. Implants that resemble the human elbow in appearance do not replicate normal behavior consistently, whereas other implants that do not resemble the human elbow closely do not deviate markedly from human behavior. Thus, much basic information about elbow form and function is needed to improve the performance of total elbow prostheses.

Ligamentous repair and reconstruction for posterolateral rotatory instability of the elbow.

We describe the intermediate results of lateral ligamentous repair or reconstruction for posterolateral rotatory instability of the elbow. Between 1986 and 1999, we performed 12 direct repairs and 33 ligament reconstructions with a tendon autograft. One patient was lost to follow-up and 44 were retrospectively studied at a mean of six years (2 to 15). Surgery restored stability in all except five patients. In two the elbow became stable after a second procedure. The mean post-operative Mayo elbow performance score was 85 points (60 to 100). The result was classified as excellent in 19, good in 13, fair in seven and poor in five patients. Thirty-eight patients (86%) were subjectively satisfied with the outcome of the operation. Better results were obtained in patients with a post-traumatic aetiology (p = 0.03), those with subjective symptoms of instability at presentation (p = 0.006) and those who had an augmented reconstruction using a tendon graft (p = 0.04). Reconstruction using a tendon graft seems to provide better results than ligament repair and the results do not seem to deteriorate with time. The outcome of this procedure is less predictable in patients with no subjective instability.

The kinematic importance of radial neck length in radial head replacement.

Comminuted radial head fractures can be treated with a radial head implant. The effects of lengthening (2.5 mm, 5 mm) and shortening (2.5 mm, 5 mm) of the radial neck, were compared to the nominal length in six human upper extremity cadavers. Total varus-valgus laxity and ulnar rotation were recorded. We hypothesized that restoring the exact length of the radius is important to maintain normal kinematics in the elbow joint. Lengthening or shortening of more than 2.5 mm significantly changed elbow kinematics. Lengthening caused a significant decrease (p < 0.001) in varus-valgus laxity, with the ulna tracking in varus and external rotation. Shortening caused a significant increase in varus-valgus laxity (p < 0.001) and ulnar rotation (p < 0.001), with the ulna tracking in valgus and internal rotation. Our study suggests that a restoration of radial length is important and that axial understuffing or overstuffing the radiohumeral joint by 2.5 mm or more, will alter elbow kinematics.

Animal models for cartilage reconstruction.

Animal models are widely used to develop and evaluate tissue-engineering techniques for the reconstruction of damaged human articular cartilage. For the purpose of this review, these model systems will include in vitro culture of animal cells and explants, heterotopic models of chondrogenesis, and articular cartilage defect models. The objectives for these preclinical studies are to engineer articular cartilage for the functional restoration of a joint surface that appears anatomically, histologically, biologically, biochemically, and mechanically to resemble the original joint surface. While no animal model permits direct application to humans, each is capable of yielding principles on which decisions can be made that might eventually translate into a human application. Clearly, the use of animal models has and will continue to play a significant role in the advancement of this field. Each animal model has specific advantages and disadvantages. The key issue in the selection of an appropriate animal model is to match the model to the question being investigated and the hypothesis to be tested. The purpose of this review is to discuss issues regarding animal model selection, the benefits and limitations of these model systems, scaffold selection with emphasis on polymers, and evaluation of the tissue-engineered articular cartilage.

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.

Dynamic pressure transmission through agarose gels.

In biomedical research, agarose gel is widely used in tissue culture systems because it permits growing cells and tissues in a three-dimensional suspension. This is especially important in the application of tissue engineering concepts to cartilage repair because it supports the cartilage phenotype. Mechanical loading, especially compression, plays a fundamental role in the development and repair of cartilage. It would be advantageous to develop a system where cells and tissues could be subjected to compression so that their responses can be studied. There is currently no information on the pressure response of agarose gel when pressure is applied to the gas phase of a culture system. To understand the transmission of pressure through the gel, we set up an apparatus that would mimic an agarose suspension tissue culture system. This consisted of a sealed metal cylinder containing air as well as a layer of agarose submerged in culture medium. Pressure responses were recorded in the air, fluid, gel center, and gel periphery using various frequencies, pressures, gel volumes, and viscosities. Regression analyses show an almost perfect linear relation between gas and gel pressures (r(2) = 0.99987, p < 0.0001, f(x) = 0.9982 x - 0.0286). The pressure transmission was complete and immediate, throughout the range of the applied pressures, frequencies, volumes, and viscosities tested. Applying dynamic pressure to the gas phase results in reproducible pressure in the agarose and, therefore, validates the use of agarose tissue culture systems in studies employing dynamic pressurization in cartilage tissue engineering.

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.

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

Periosteal transplantation is being used clinically to repair articular defects. Isolated cells and very small periosteal explants can be grown in tissue culture, but it will be necessary to test larger sizes for tissue engineering to be applied to clinical transplantation of periosteum. This study was conducted to assess the chondrogenic potential of different sizes of periosteal explants in agarose culture. Ninety-six rabbit tibial periosteal explants in three different sizes (small 1.5 x 2, medium 3 x 2, and large 4 x 6 mm, 32 pieces per size) were cultured in agarose suspension for 6 wk and given TGF-beta1 (10 ng/mL) for the first 2 wk. Tissue growth, as indicated by normalized final wet weights of the explants after 6 wk in culture, was inversely proportional to explant size. Cartilage formation was observed in all explants. Histomorphometry revealed that cartilage formation was significantly better for the smaller explants (80% cartilage), but similar in the medium and larger explants (60% cartilage). Similar proportions of type II collagen were present in the different-sized explants. This study demonstrates that various sizes of periosteal explants can be grown in culture. Abundant cartilage was produced even by the large explants.

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.

Effect of pregnancy on joint contracture in the rat knee.

As there is evidence that ligamentous laxity is affected by the female hormones, we hypothesized that hormonal changes occurring during pregnancy could have a therapeutic role in preventing the development of a joint contracture. Knee joint contractures were created in pregnant and nonpregnant rats. After 2 wk of immobilization, the degree of contracture was measured with structural properties of the medial collateral and anterior cruciate ligaments and the pubic symphysis. Although not statistically significant, there was a general trend toward reduced contracture in pregnant compared with nonpregnant rats. Cutting the posterior capsule significantly decreased contracture for both the pregnant and nonpregnant groups, confirming the contribution of capsular structures to contracture. Ultimate loads of the medial collateral and anterior cruciate ligaments significantly decreased after immobilization compared with control, but there was no significant effect due to pregnancy. Stiffness and ultimate load of the pubic symphysis were not significantly different between pregnant and nonpregnant groups. The trend toward reduced contracture with pregnancy points toward a possible therapeutic role for female hormones in the prevention of postoperative and/or posttraumatic joint contracture.

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.

An effective method of completely removing contaminating genomic DNA from an RNA sample to be used for PCR.

A simple method for removing contaminating genomic DNA from an RNA preparation is presented. The method involves digestion of the RNA with RNase-free DNase I at room temperature followed by inactivation of the enzyme at 65 degrees C in presence of EDTA. This method produces an RNA sample that is negative for genomic DNA by PCR.

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.