James S. Fitzsimmons

The role of periosteum in cartilage repair.

Periosteum, which can be grown in cell and whole tissue cultures, may meet one or more of the three prerequisites for tissue engineered cartilage repair. Periosteum contains pluripotential mesenchymal stem cells with the potential to form either cartilage or bone. Because it can be transplanted as a whole tissue, it can serve as its own scaffold or a matrix onto which other cells and/or growth factors can be adhered. Finally, it produces bioactive factors that are known to be chondrogenic. The chondrocyte precursor cells reside in the cambium layer. These vary in total density and volume with age and in different donor sites. The advantages of whole tissue periosteal transplants for cartilage repair include the fact that this tissue meets the three primary requirements for tissue engineering: a source of cells, a scaffold for delivering and retaining them, and a source of local growth factors. Many growth factors that regulate chondrocytes and cartilage development are synthesized by periosteum in conditions conducive to chondrogenesis. These include transforming growth factor-beta 1, insulinlike growth factor-1, growth and differentiation factor-5, bone morphogenetic protein-2, integrins, and the receptors for these molecules. By additional study of the molecular events in periosteal chondrogenesis, it may be possible to optimize its capacity for articular cartilage repair.

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.

Brief exposure to high-dose transforming growth factor-β1 enhances periosteal chondrogenesis in vitro: A preliminary report

Background: Articular cartilage has limited potential for repair. There have been various attempts aimed at improving the repair process in articular cartilage. Transforming growth factor-&bgr;1 (TGF-&bgr;1) has a stimulatory effect on chondrogenesis in periosteal explants. The purpose of the present study was to determine the effect of brief exposures (i.e., thirty and sixty minutes) of high concentrations of TGF-&bgr;1 on periosteal chondrogenesis. Methods: Five hundred and seventy-three periosteal explants were harvested from forty-six two-month-old male New Zealand White rabbits. Explants were exposed to 50 or 100 ng/mL of TGF-&bgr;1 for thirty or sixty minutes. The amount of cartilage formed was then determined with use of a standardized six-week agarose culture assay. Results: There was a significant increase in the amount of cartilage formation (p < 0.01), Type-II collagen content (p < 0.05), and sulfate incorporation (p < 0.0001) in explants treated with TGF-&bgr;1. Maximal stimulation occurred following exposure to 100 ng/mL of TGF-&bgr;1 for thirty minutes. There was also an increase in chondrocyte proliferation as measured by [3 H-] thymidine incorporation on day 5 of culture (p < 0.049). Conclusions: The findings of this study indicate that exposure to TGF-&bgr;1 has a stimulatory effect on periosteal chondrogenesis. This stimulatory effect is observed even with a very brief exposure time of thirty minutes. Clinical Relevance: A possible clinical application of these findings is exposure of periosteal grafts that are currently utilized clinically to resurface articular defects to TGF-&bgr;1 during the short time between graft procurement and implantation into the joint. This may obviate the need for intra-articular administration of TGF-&bgr;1 and may enhance the ultimate graft incorporation and quality of cartilage repair.

Enhancement of periosteal chondrogenesis in vitro. Dose-response for transforming growth factor-beta 1 (TGF-beta 1).

Transforming growth factor-beta 1 (TGF-beta 1) has been shown to stimulate chondrogenesis in periosteal explants cultured in agarose suspension. In this study, the dose-response curve for such enhancement was measured. Periosteal explants and fascia lata were harvested from two-month-old rabbits, cultured for six weeks with 0, 0.1, 1, 5, 10, 50, or 100 ng/mL TGF-beta 1 in agarose suspension, then analyzed by histomorphometry and quantitative collagen typing. Cartilage was produced by seven of 11 (64%) of the control periosteal explants cultured in agarose suspension without TGF-beta 1. Transforming growth factor-beta 1 enhanced chondrogenesis in a dose-dependent manner in the range 0.1-100 ng/mL. It was most effective at 50 ng/mL. At very high doses (50 and 100 ng/mL) of TGF-beta 1, even fascia lata control explants exhibited chondrogenesis. These data indicate that TGF-beta 1 can induce differentiation toward cartilage production as well as enhance it once it has been initiated.

The importance of procedure specific training in harvesting periosteum for chondrogenesis

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

Tissue engineering of cartilage using poly-ɛ-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.

The effect of scaffold composition on the early structural characteristics of chondrocytes and expression of adhesion molecules

Previously we demonstrated that chondrocyte ECM synthesis and mitotic activity was dependent on scaffold composition when cultured on uncoated PCL scaffolds (pPCL) or PCL composites containing hyaluronan (PCL/HA), chitosan (PCL/CS), fibrin (PCL/F), or collagen type I (PCL/COL1). We hypothesized that initial cell contact with these biomaterials results in ultrastructural changes and alters CD44 and integrin beta1 expression. The current study was designed to investigate the early ultrastructural responses of chondrocytes on these scaffolds and expression of CD44 and integrin beta1. A common observation 1 h after seeding was the abundance of cell processes. Different types of cell processes occurred in different areas of the same cell and on different cells within the same composite. Chondrocytes seeded onto PCL/CS had the greatest cell surface enhancement. PCL/HA promoted CD44 expression and almost spherical cells with a low degree of surface enhancement. PCL/COL1 enabled continuing expression of integrin beta1 and CD44. In contrast, cells in PCL/CS, PCL/F and pPCL promoted elliptic cells with a higher degree of surface enhancement and no prolonged CD44 and integrin beta1 expression. A strong variability of cell surface processes indicated either reparative or degenerative adaptation to the artificial environment. Interestingly, we found initial integrin beta1 expression in all composite scaffolds, but not in pPCL although this promoted strong adhesiveness as indicated by the formation of stress fibers. In conclusion, chondrocytes respond to biomaterials early after implantation by altering ultrastructural characteristics and expression of CD44 and integrin beta1.

The biomechanical effect of prosthetic design on radiocapitellar stability in a terrible triad model.

Objectives: The integrity of elbow soft tissues affects radiocapitellar joint stability in the presence of bipolar radial head (RH) prostheses. This study examined the effect on radiocapitellar stability of monopolar designs versus bipolar RH prostheses in an elbow model with a surgically controlled terrible triad injury. Methods: In each of 8 fresh-frozen elbow specimens (4 male and 4 female), a terrible triad fracture dislocation was created through soft tissue releases, coronoid fracture, and RH resection. Radiocapitellar stability was recorded under the following 3 sets of conditions: (1) surgical control (native RH), (2) RH replacement (circular monopolar or bipolar), (3) replacement with alternate circular RH not used in condition 2, and (4) replacement with the anatomic RH. Results: The type of RH used significantly impacted the mean peak force required to resist posterior subluxation (p = 0.0001). The mean peak subluxation force of the bipolar prosthesis (1 ± 1 N) was significantly less than both the anatomic (16 ± 1 N) and nonanatomic circular (12 ± 1 N) implants (p = 0.0002). The peak subluxation force of the native RH (18 ± 2 N) was not different than the anatomic implant (p = 0.09) but was greater than the nonanatomic circular design (p = 0.0006). Conclusions: Monopolar RHs confer greater radiocapitellar stability than bipolar implants in the setting of terrible triad injuries. Of the 2 monopolar designs tested, the anatomic design provided more stability than the non-anatomic RH prosthesis.

Stress Shielding Around Radial Head Prostheses

PURPOSE Stress shielding is known to occur around rigidly fixed implants. We hypothesized that stress shielding around radial head prostheses is common but nonprogressive. In this study, we present a classification scheme to support our radiographic observations. METHODS We reviewed charts and radiographs of 86 cases from 79 patients with radial head implants from both primary and revision surgeries between 1999 and 2009. Exclusion criteria included infection, loosening, or follow-up of less than 12 months. We classified stress shielding as: I, cortical thinning; II, partially (IIa) or circumferentially (IIb) exposed stem; and III, impending mechanical failure. RESULTS Of 26 well-fixed stems, 17 (63%) demonstrated stress shielding: I = 2, II = 15 (IIa = 12, IIb = 3), and III = 0. We saw stress shielding with all stem types: cemented or noncemented; long or short; and straight, curved, or tapered. The only significant difference was that stems implanted into the radial shaft had less stress shielding than stems implanted into the neck or tuberosity (P = .03). The average follow-up was 33 months (range, 13-70 mo). Stress shielding was detectable by an average of 11 months (range, 1-15 mo). The pattern of bone loss was similar in 16 of 17 cases (94%), starting on the outer periosteal cortex. The 3 cases with circumferential exposure of the stem (stage IIb) averaged 2.6 mm (range, 1-4 mm) of exposed stem. Stress shielding never extended to the bicipital tuberosity, and there were no cases of impending mechanical failure. CONCLUSIONS Stress shielding around radial head prostheses is common, regardless of stem design. However, it is typically minor, nonprogressive, and of questionable clinical consequence. TYPE OF STUDY/LEVEL OF EVIDENCE Therapeutic IV.

Validation of a photography-based goniometry method for measuring joint range of motion.

BACKGROUND A critical component of evaluating the outcomes after surgery to restore lost elbow motion is the range of motion (ROM) of the elbow. This study examined if digital photography-based goniometry is as accurate and reliable as clinical goniometry for measuring elbow ROM. MATERIALS AND METHODS Instrument validity and reliability for photography-based goniometry were evaluated for a consecutive series of 50 elbow contractures by 4 observers with different levels of elbow experience. Goniometric ROM measurements were taken with the elbows in full extension and full flexion directly in the clinic (once) and from digital photographs (twice in a blinded random manner). RESULTS Instrument validity for photography-based goniometry was extremely high (intraclass correlation coefficient: extension = 0.98, flexion = 0.96). For extension and flexion measurements by the expert surgeon, systematic error was negligible (0° and 1°, respectively). Limits of agreement were 7° (95% confidence interval [CI], 5° to 9°) and -7° (95% CI, -5° to -9°) for extension and 8° (95% CI, 6° to 10°) and -7° (95% CI, -5° to -9°) for flexion. Interobserver reliability for photography-based goniometry was better than that for clinical goniometry. The least experienced observers photographic goniometry measurements were closer to the reference measurements than the clinical goniometry measurements. CONCLUSIONS Photography-based goniometry is accurate and reliable for measuring elbow ROM. The photography-based method relied less on observer expertise than clinical goniometry. This validates an objective measure of patient outcome without requiring doctor-patient contact at a tertiary care center, where most contracture surgeries are done.