A. Simon Turner

Anabolism: Low mechanical signals strengthen long bones

Although the skeletons adaptability to load-bearing has been recognized for over a century, the specific mechanical components responsible for strengthening it have not been identified. Here we show that after mechanically stimulating the hindlimbs of adult sheep on a daily basis for a year with 20-minute bursts of very-low-magnitude, high-frequency vibration, the density of the spongy (trabecular) bone in the proximal femur is significantly increased (by 34.2%) compared to controls. As the strain levels generated by this treatment are three orders of magnitude below those that damage bone tissue, this anabolic, non-invasive stimulus may have potential for treating skeletal conditions such as osteoporosis.

Quantity and Quality of Trabecular Bone in the Femur Are Enhanced by a Strongly Anabolic, Noninvasive Mechanical Intervention

The skeletons sensitivity to mechanical stimuli represents a critical determinant of bone mass and morphology. We have proposed that the extremely low level (<10 microstrain), high frequency (20‐50 Hz) mechanical strains, continually present during even subtle activities such as standing are as important to defining the skeleton as the larger strains typically associated with vigorous activity (>2000 microstrain). If these low‐level strains are indeed anabolic, then this sensitivity could serve as the basis for a biomechanically based intervention for osteoporosis. To evaluate this hypothesis, the hindlimbs of adult female sheep were stimulated for 20 minutes/day using a noninvasive 0.3g vertical oscillation sufficient to induce approximately 5 microstrain on the cortex of the tibia. After 1 year of stimulation, the physical properties of 10‐mm cubes of trabecular bone from the distal femoral condyle of experimental animals (n = 8) were compared with controls (n = 9), as evaluated using microcomputed tomography (μCT) scanning and materials testing. Bone mineral content (BMC) was 10.6% greater (p < 0.05), and the trabecular number (Tb.N) was 8.3% higher in the experimental animals (p < 0.01), and trabecular spacing decreased by 11.3% (p < 0.01), indicating that bone quantity was increased both by the creation of new trabeculae and the thickening of existing trabeculae. The trabecular bone pattern factor (TBPf) decreased 24.2% (p < 0.03), indicating trabecular morphology adapting from rod shape to plate shape. Significant increases in stiffness and strength were observed in the longitudinal direction (12.1% and 26.7%, respectively; both, p < 0.05), indicating that the adaptation occurred primarily in the plane of weightbearing. These results show that extremely low level mechanical stimuli improve both the quantity and the quality of trabecular bone. That these deformations are several orders of magnitude below those peak strains which arise during vigorous activity indicates that this biomechanically based signal may serve as an effective intervention for osteoporosis.

Biologic augmentation of rotator cuff tendon-healing with use of a mixture of osteoinductive growth factors.

BACKGROUND Clinical studies have demonstrated a high rate of incomplete healing of rotator cuff tendon repair. Since healing of such a repair is dependent on bone ingrowth into the repaired tendon, we hypothesized that osteoinductive growth factors would improve rotator cuff tendon-healing. METHODS Seventy-two skeletally mature sheep underwent detachment of the infraspinatus tendon followed by immediate repair. The animals received one of three treatments at the tendon-bone interface: (1) an osteoinductive bone protein extract on a Type-I collagen sponge carrier, (2) the collagen sponge carrier alone, and (3) no implant. The animals were killed at six and twelve weeks, and the repaired rotator cuff was evaluated with use of magnetic resonance imaging, plain radiographs, histologic analysis, and biomechanical testing. RESULTS A gap consistently formed between the end of the repaired tendon and bone in this model, with reparative scar tissue and new bone spanning the gap. Magnetic resonance imaging showed that the volume of newly formed bone (p < 0.05) and soft tissue (p < 0.05) in the tendon-bone gap were greater in the growth factor-treated animals compared with the collagen sponge control group at both time-points. Histologic analysis showed a fibrovascular tissue in the interface between tendon and bone, with a more robust fibrocartilage zone between the bone and the tendon in the growth factor-treated animals. The repairs that were treated with the osteoinductive growth factors had significantly greater failure loads at six weeks and twelve weeks (p < 0.05); however, when the data were normalized by tissue volume, there were no differences between the groups, suggesting that the treatment with growth factor results in the formation of poor-quality scar tissue rather than true tissue regeneration. The repairs that were treated with the collagen sponge carrier alone had significantly greater stiffness than the growth factor-treated group at twelve weeks (p = 0.005). CONCLUSIONS This model tests the effects of growth factors on scar tissue formation in a gap between tendon and bone. The administration of osteoinductive growth factors resulted in greater formation of new bone, fibrocartilage, and soft tissue, with a concomitant increase in tendon attachment strength but less stiffness than repairs treated with the collagen sponge carrier alone.

rhBMP-12 Accelerates Healing of Rotator Cuff Repairs in a Sheep Model

BACKGROUND The success rate of rotator cuff repairs is variable. This study was performed to evaluate the ability of recombinant human bone morphogenetic protein-12 (rhBMP-12), administered in several carriers, to accelerate healing in a sheep model of rotator cuff repair. METHODS Local retention of tracer amounts of radiolabeled rhBMP-12, added to non-radiolabeled rhBMP-12 delivered in buffer, hyaluronan paste or sponges, or Type-I or Type-I/III collagen sponges was first evaluated with use of gamma scintigraphy in a pilot study of a rat intramuscular implant model. The rhBMP-12/paste and sponge combinations were then evaluated in eight sheep each with unilateral complete detachment and subsequent double-row reattachment of the infraspinatus tendon to the proximal part of the humerus. Contralateral, normal shoulders from sixteen sheep and shoulders in which a repair had been done without administration of rhBMP-12 in fourteen sheep were also evaluated. The rhBMP-12/Type-I and Type-I/III collagen sponge combinations were each evaluated in eight additional sheep on the basis of superior efficacy. The Type-I/III collagen sponge alone was evaluated in ten sheep to examine the effect of a collagen carrier. Ultrasound imaging was performed at four and eight weeks. Radiographic evaluation, mechanical testing, and biochemical evaluation were performed at eight weeks. Histological evaluation was performed on specimens from the sites of selected repairs following mechanical testing. RESULTS The sponge carriers had longer local retention of rhBMP-12 than did the buffer or paste carriers in the rat models. All of the sheep shoulder-repair groups demonstrated ultrasound evidence of a gap between the tendon and the humeral insertion. The gap length and the cross-sectional area of the repair tissue decreased with time. The mechanical properties of the repairs treated with rhBMP-12 and hyaluronan paste were similar to those of the untreated repairs. The maximum loads for the rhBMP-12/hyaluronan sponge and rhBMP-12/collagen sponge-treated repairs were 2.1 and 2.7 times greater, respectively, than the loads for the untreated repairs and were 33% and 42% of the value for the normal tendon at eight weeks. The maximum loads for the repairs treated with rhBMP-12 and a Type-I or Type-I/III collagen sponge were 2.1 times greater than those for the repairs treated with the Type-I/III collagen sponge alone. Changes in maximum stiffness followed a similar pattern. Histological evaluation demonstrated accelerated healing of the rhBMP-12-treated repairs compared with the untreated repairs. Bone formation was observed in all repairs, and biochemical measurements were not equivalent to those of normal tendon at eight weeks. CONCLUSIONS Delivery of rhBMP-12 in a collagen or hyaluronan sponge resulted in accelerated healing of acute full-thickness rotator cuff repairs in a sheep model. CLINICAL RELEVANCE Delivery of rhBMP-12 in several sponge carriers has the potential to accelerate healing of rotator cuff repairs. Accelerated repair may allow shorter rehabilitation and an earlier return to occupational and recreational activities.

Histologic evaluation of the efficacy of rhBMP-2 compared with autograft bone in sheep spinal anterior interbody fusion.

Study Design. The sheep anterior lumbar spinal fusion model was used to study the efficacy of recombinant human bone morphogenetic protein-2 (rhBMP-2)–collagen composite in comparison with autograft to enhance spinal interbody fusion. Comparisons were drawn from temporal radiographic and end-point biomechanical and histologic data. Objective. To analyze histologically the ability of rhBMP-2 to achieve complete arthrodesis between vertebral bodies. Summary of Background Data. Studies using rhBMP for enhancement of anterior interbody fusion have used numerous endpoints. However, systematic histologic evaluation of the fusion has not been conducted. Methods. Twelve sheep underwent single-level anterior lumbar interbody fusion performed with a cylindrical fenestrated titanium interbody fusion device (INTER FIX, Medtronic Sofamor Danek, Inc., Memphis, TN). The device was filled either with rhBMP-2–collagen (n = 6) or autogenous iliac crest bone graft (n = 6). Radiologic evaluation was carried out at 2-month intervals, and all sheep were killed 6 months after surgery. Nondestructive biomechanical testing for stiffness to flexion, extension, and lateral bending moments, un-decalcified histology, and qualitative and quantitative histologic evaluation were performed. Results. Radiographs revealed a bony bridge anterior to the cage in five of six rhBMP-2-treated animals, whereas it was present only in one of five in the autogenous bone graft group. Segments treated with rhBMP-2 were 20% stiffer in flexion than autograft-treated segments at 6 months. Six of six in the rhBMP-2 group and two of six in the autograft group showed complete fusion. There was a significantly higher rate of bony continuity observed at the fenestrations of the rhBMP-2 group. Three times more number of cage fenestrations in the rhBMP-2 group demonstrated “all-bone” when compared with the autograft group (P < 0.001). Further, the scar tissue in and around the autograft-treated cages was 16-fold more (P < 0.01) than that seen for rhBMP-2-treated cages. Conclusions. The study demonstrates that rhBMP-2 can lead to earlier radiologic fusion and a more consistent increased stiffness of the segments when compared with autograft in sheep anterior lumbar interbody fusion. Furthermore, a three times higher histologic fusion rate is attainable with significantly reduced fibrous tissue around the implant when rhBMP-2 is used.

The Effects of Augmentation With Swine Small Intestine Submucosa on Tendon Healing Under Tension Histologic and Mechanical Evaluations in Sheep

Background Rotator cuff failure after surgery may be attributed to inferior tissue healing properties that result from repetitive cyclic loading during early rehabilitation. Enhancing the biological healing process may reduce the incidence of failures after rotator cuff repairs. Hypothesis Augmentation of rotator cuff tissue using swine small intestine submucosa in a sheep model will improve the rate and quality of tissue repair. Study Design Controlled laboratory study. Methods We resected and reattached 26 sheep infraspinatus tendons under tension, with 13 animals receiving a small intestine submucosa patch (augmented group). Animals were sacrificed at 12 weeks, and biomechanical testing and histologic evaluation were performed. Biomechanical testing was completed in 10 tendons from each group. Specimens were loaded to failure at a constant displacement to obtain the load deformation curve used to calculate load to failure and stiffness of the healed bone-tendon interface. Histologic testing addressed tissue healing at the bone-tendon interface. Results The load-to-failure data did not indicate a significant difference between the augmented and nonaugmented groups (1252 ± 402 N vs 985 ± 459 N, respectively; P >. 05). However, the augmented group had significantly better stiffness than the nonaugmented group (215 ± 44 N/mm vs 154 ± 63 N/mm, respectively; P =. 03). Histologic data revealed that the infraspinatus tendon in all specimens inserted into the bone through a zone of fibrocartilage, although none of the patches were intact. Conclusion Although there were no differences in the load-to-failure data between the 2 groups, the statistically significant improvement in stiffness for the augmented group is clinically relevant. Stiffness is the biomechanical parameter representing the tissue response to subdestructive loads seen with early rehabilitation. Augmenting the repair with a collagen matrix improved the early healing characteristics of the repair construct. Clinical Relevance Enhancing the biological process of tendon healing under tension by using a collagen matrix patch may improve the ultimate success of rotator cuff repair.

Meniscal Allograft Transplantation in the Sheep Knee Evaluation of Chondroprotective Effects

Background Early protection of articular cartilage, before degenerative changes appear on radiographs, should result in better long-term results, but scientific evidence regarding the effectiveness of meniscal transplantation is lacking. Purpose To assess the chondroprotective effects of a new meniscal allograft transplantation animal model and evaluate a magnetic resonance imaging parameter, T2 mapping, in articular cartilage after meniscectomy and meniscal transplantation. Study Design Controlled laboratory study. Methods Forty-five skeletally mature sheep were placed into 3 surgical groups lateral meniscectomy (n = 24), meniscal allograft transplant (n = 17), and sham (n = 4). Animals were sacrificed at 2, 4, or 12 months. Cartilage was assessed by gross inspection, magnetic resonance imaging, T2 mapping, biomechanical testing, and semiquantitative histologic analysis. Results There were no differences between the sham operation and nonoperated control limbs. Compared with control limbs, meniscectomy resulted in significant increases in cartilage degeneration by all objective criteria (P < .01). Compared with meniscectomy, meniscal allograft transplantation resulted in significant decreases in cartilage degeneration (P < .02). There were significant correlations between T2-mapping data and all other traditional outcomes measures (P < .05, r2 = 0.37-0.67). Compared with the nonoperated control limbs, allograft transplants demonstrated no significant differences at 2 months in any category, except magnetic resonance imaging data. By 4 months, nonoperated control limbs demonstrated significantly less wear compared to allograft limbs in all categories except modified Mankin scores. Conclusion This model demonstrated significant chondroprotection compared with meniscectomy but demonstrated more cartilage wear at 4 months compared to control limbs. A high degree of allograft cell viability and vascular ingrowth was seen in allograft explants. T2 mapping may provide an accurate noninvasive measure of early cartilage degeneration after meniscectomy, as well as cartilage protection after meniscal allograft transplantation. Clinical Relevance This study establishes a reliable animal model for meniscal allograft transplantation and provides evidence for the utility of T2 mapping at clinically relevant magnetic resonance imaging field strengths for evaluation of early cartilage degeneration.

Augmentation of a Rotator Cuff Suture Repair Using rhPDGF-BB and a Type I Bovine Collagen Matrix in an Ovine Model

Background Rotator cuff tears are a common source of shoulder pain. High rates (20%-94%) of structural failure of the repair have been attributed to multiple factors, including poor repair tissue quality and tendon-to-bone integration. Biologic augmentation using growth factors has potential to promote tendon-to-bone integration, improving the function and long-term success of the repair. One such growth factor is platelet-derived growth factor–BB (PDGF-BB), which has been shown to improve healing in tendon and bone repair models. Hypothesis Recombinant human PDGF-BB (rhPDGF-BB) combined with a highly porous type I bovine collagen matrix will improve the biomechanical function and morphologic appearance of the repair in a dose-dependent manner, relative to a suture-only control, after 12 weeks in an acute ovine model of rotator cuff repair. Study Design Controlled laboratory study. Methods An interpositional graft consisting of rhPDGF-BB and a type I collagen matrix was implanted in an ovine model of rotator cuff repair. Biomechanical and histologic analyses were performed to determine the functional and anatomic characteristics of the repair after 12 weeks. Results A significant increase in the ultimate load to failure was observed in repairs treated with 75 μg (1490.5 ± 224.5 N, P = .029) or 150 μg (1486.6 ± 229.0 N, P = .029) of rhPDGF-BB, relative to suture-only controls (910.4 ± 156.1 N) and the 500-μg rhPDGF-BB group (677.8 ± 105.9 N). The 75-μg and 150-μg rhPDGF-BB groups also exhibited increased tendon-to-bone inter-digitation histologically. No differences in inflammation or cellularity were observed among treatments. Conclusion This study demonstrated that an interpositional graft consisting of rhPDGF-BB (75 or 150 μg) and a type I collagen matrix was able to improve the biomechanical strength and anatomic appearance in an ovine model of rotator cuff repair compared to a suture-only control and the 500-μg rhPDGF-BB group. Clinical Relevance Recombinant human PDGF-BB combined with a type I collagen matrix has potential to be used to augment surgical repair of rotator cuff tears, thereby improving clinical success.

Biochemical effects of estrogen on articular cartilage in ovariectomized sheep

Cartilage is a sex-hormone-sensitive tissue but the role of estrogen in the pathogenesis of osteoarthritis (OA) remains controversial. In this study, intrinsic material properties and thickness of articular cartilage of the knee joint of ovariectomized (OVX) and estrogen-treated sheep were measured. Skeletally mature ewes (N = 36, same breed, same housing 4-5 years old) were divided into; sham treated (n = 9), OVX (N = 13), OVX plus one estradiol implant (OVXE; N = 10) and OVX plus two estradiol implants (OVX2E; N = 4). Twelve months following sham procedure or OVX, sheep were euthanized and articular cartilage from a total of 216 points in the left femorotibial (knee) joints was tested for aggregate modulus, Poissons ratio, permeability, thickness and shear modulus (six sites per sheep). When all of the sites in each knee were grouped together, OVX had a significant effect on articular cartilage. The sham cartilage of all sites grouped together had a larger aggregate modulus (P = 0.001) and a larger shear modulus (P = 0.054) than the OVX tissue. No statistically significant differences were seen for permeability and thickness between OVX, sham, OVXE and OVX2E. Differences existed in biomechanical properties at the different sites that were tested. Overall, no one location tended to be lowest or highest for all variables. This biomechanical study suggests that OVX may have a detrimental effect on the intrinsic material properties of the articular cartilage of the knee, even though the cartilage of the OVX animals appeared normal. Treatment with estradiol implants ameliorated these deleterious effects and may have helped maintain the tissues structural integrity. Our study supports epidemiological studies of OA in women after menopause. The protective effect of estrogen and its therapeutic effect remain to be further defined. This model may allow the relationship of estrogen and estrogen antagonists to be studied in greater detail, and may be valuable for the study of the pathogenesis and therapies of OA of postmenopausal women, particularly in its early stages.

Hydrogel Meniscal Replacement in the Sheep Knee: Preliminary Evaluation of Chondroprotective Effects

Background Meniscal allograft transplantation has become a viable surgical alternative for a select group of patients with deficient or irreparable menisci. Subjective results are encouraging; long-term success, durability, and safety of allograft meniscal transplantation are uncertain. Purposes To evaluate a novel hydrogel meniscal replacement implant in an ovine model and assess chondroprotective effects of this hydrogel meniscal replacement using several validated outcome measures. Study Design Controlled laboratory study. Methods Fourteen skeletally mature sheep underwent hydrogel meniscal replacement; 45 additional animals had previously undergone 1 of 3 operations: lateral meniscectomy (24), meniscal allograft transplant (17), and sham (4). Animals were sacrificed at 2, 4, or 12 months. Cartilage was assessed by magnetic resonance imaging, gross inspection, biomechanical testing, and semiquantitative histological analysis. Results There were no differences between the sham operation and nonoperated control limbs. Compared with meniscectomy, hydrogel meniscal replacement resulted in significantly decreased cartilage degeneration with all outcome parameters (P < .05). Compared with nonoperated control limbs, hydrogel meniscal replacements demonstrated no significant differences at 2 months in any category. By 4 months, hydrogel limbs demonstrated significantly greater cartilage degeneration than did nonoperated control limbs in all categories. Compared with meniscal allograft transplantation animals, hydrogel meniscal replacements demonstrated no differences at 2 months but had significantly increased cartilage degeneration in the peripheral zone of the tibial plateau at 4 months (P < .05). At 1 year, all hydrogel implants had developed complete radial splits in the posterior third of the implant. Conclusion Although promising preliminary results for hydrogel meniscal replacement were seen at early time points, significant cartilage degeneration and implant failure were seen at 1 year, and overall performance was worse than was allograft transplantation. Improvements in hydrogel material properties and surface characteristics and more accurate size matching may improve outcomes. Clinical Relevance Improvements in the hydrogel material properties and surface characteristics and more accurate size matching may lead to the use of hydrogel implants in humans.