Michael D. Brodt

The effect of gap formation at the repair site on the strength and excursion of intrasynovial flexor tendons. An experimental study on the early stages of tendon-healing in dogs.

BACKGROUND Elongation (gap formation) at the repair site has been associated with the formation of adhesions and a poor functional outcome after repair of flexor tendons. Our objectives were to evaluate the prevalence of gap formation in a clinically relevant canine model and to assess the effect of gap size on the range of motion of the digits and the mechanical properties of the tendons. METHODS We performed operative repairs after sharp transection of sixty-four flexor tendons in thirty-two adult dogs. Rehabilitation with passive motion was performed daily until the dogs were killed at ten, twenty-one, or forty-two days postoperatively. Eight tendons ruptured in vivo. In the fifty-six intact specimens, the change in the angles of the proximal and distal interphalangeal joints and the linear excursion of the flexor tendon were measured as a 1.5-newton force was applied to the tendon. The gap at the repair site was then measured, and the isolated tendons were tested to failure in tension. RESULTS Twenty-nine tendons had a gap of less than one millimeter, twelve had a gap of one to three millimeters, and fifteen had a gap of more than three millimeters. Neither the time after the repair nor the size of the gap was found to have a significant effect on motion parameters (p > 0.05); however, the ultimate force, repair-site rigidity, and repair-site strain at twenty newtons were significantly affected by these parameters (p < 0.05). Testing of the tendons with a gap of three millimeters or less revealed that, compared with the ten-day specimens, the forty-two-day specimens failed at a significantly (90 percent) higher force (p < 0.01) and had a significantly (320 percent) increased rigidity (p < 0.01) and a significantly (60 percent) decreased strain at twenty newtons (p < 0.05). In contrast, the tensile properties of the tendons that had a gap of more than three millimeters did not change significantly with time. CONCLUSIONS Our data indicate that, in a dog model involving sharp transection followed by repair, a gap at the repair site of more than three millimeters does not increase the prevalence of adhesions or impair the range of motion but does prevent the accrual of strength and stiffness that normally occurs with time.

SHIP-deficient mice are severely osteoporotic due to increased numbers of hyper-resorptive osteoclasts

The hematopoietic-restricted protein Src homology 2–containing inositol-5-phosphatase (SHIP) blunts phosphatidylinositol-3-kinase-initiated signaling by dephosphorylating its major substrate, phosphatidylinositol-3,4,5-trisphosphate. As SHIP−/− mice contain increased numbers of osteoclast precursors, that is, macrophages, we examined bones from these animals and found that osteoclast number is increased two-fold. This increased number is due to the prolonged life span of these cells and to hypersensitivity of precursors to macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL). Similar to pagetic osteoclasts, SHIP−/− osteoclasts are enlarged, containing upwards of 100 nuclei, and exhibit enhanced resorptive activity. Moreover, as in Paget disease, serum levels of interleukin-6 are markedly increased in SHIP−/− mice. Consistent with accelerated resorptive activity, 3D trabecular volume fraction, trabecular thickness, number and connectivity density of SHIP−/− long bones are reduced, resulting in a 22% loss of bone-mineral density and a 49% decrease in fracture energy. Thus, SHIP negatively regulates osteoclast formation and function and the absence of this enzyme results in severe osteoporosis.

Growing C57Bl/6 Mice Increase Whole Bone Mechanical Properties by Increasing Geometric and Material Properties

In vivo murine models are becoming increasingly important in bone research. To establish baseline data for researchers using these models, we studied the long bones from C57BL/6 female mice, a strain that is widely used in bone research. We determined the femoral structural and material properties in both torsion and four‐point bending for mice at ages 4–24 weeks. Measurements of femoral cross‐sectional geometry and tibial densitometric properties were also obtained. Results indicated that all structural properties (except ultimate energy), changed significantly with age (p < 0.001). Ultimate torque, ultimate moment, torsional rigidity, and bending rigidity all increased to peak values at 20 weeks, whereas ultimate rotation and ultimate displacement decreased to minimum values at 20 weeks. Our data indicate that increases in the material properties contributed more than increases in cross‐sectional geometry to the changes in structural rigidity and ultimate load. For example, from 4–20 weeks torsional rigidity increased 1030%, while shear modulus increased 610% and the polar moment of inertia (a measure of the geometric resistance to rotation) increased by only 85%. Changes in the cross‐sectional geometry with age were due to increases in periosteal diameter and decreases in endosteal diameter. In general, both torsion and bending techniques revealed large changes in structural and material properties with age. We conclude that peak bone strength is not achieved before 20 weeks in C57BL/6 female mice, and that torsion and four‐point bending tests are equally well suited for evaluating mechanical properties of murine long bones.

Type 1 Diabetes in Young Rats Leads to Progressive Trabecular Bone Loss, Cessation of Cortical Bone Growth, and Diminished Whole Bone Strength and Fatigue Life†‡

People with diabetes have increased risk of fracture disproportionate to BMD, suggesting reduced material strength (quality). We quantified the skeletal effects of type 1 diabetes in the rat. Fischer 344 and Sprague‐Dawley rats (12 wk of age) were injected with either vehicle (Control) or streptozotocin (Diabetic). Forelimbs were scanned at 0, 4, 8, and 12 wk using pQCT. Rats were killed after 12 wk. We observed progressive osteopenia in diabetic rats. Trabecular osteopenia was caused by bone loss: volumetric BMD decreased progressively with time in diabetic rats but was constant in controls. Cortical osteopenia was caused by premature arrest of cortical expansion: cortical area did not increase after 4–8 wk in diabetic rats but continued to increase in controls. Postmortem μCT showed a 60% reduction in proximal tibial trabecular BV/TV in diabetic versus control rats, whereas moments of inertia of the ulnar and femoral diaphysis were reduced ∼30%. Monotonic bending tests indicated that ulna and femora from diabetic animals were ∼25% less stiff and strong versus controls. Estimates of material properties indicated no changes in elastic modulus or ultimate stress but modest (∼10%) declines in yield stress for diabetic bone. These changes were associated with a ∼50% increase in the nonenzymatic collagen cross‐link pentosidine. Last, cyclic testing showed diminished fatigue life in diabetic bones at the structural (force) level but not at the material (stress) level. In summary, type 1 diabetes, left untreated, causes trabecular bone loss and a reduction in diaphyseal growth. Diabetic bone has greatly increased nonenzymatic collagen cross‐links but only modestly reduced material properties. The loss of whole bone strength under both monotonic and fatigue loading is attributed mainly to reduced bone size.

Attenuated Response to In Vivo Mechanical Loading in Mice With Conditional Osteoblast Ablation of the Connexin43 Gene (Gja1)

Introduction: In vitro data suggest that gap junctional intercellular communication mediated by connexin43 (Cx43) plays an important role in bone cell response to mechanical stimulation. We tested this hypothesis in vivo in a model of genetic deficiency of the Cx43 gene (Gja1).

Decreased collagen organization and content are associated with reduced strength of demineralized and intact bone in the SAMP6 mouse.

To examine the link between bone material properties and skeletal fragility, we analyzed the mechanical, histological, biochemical, and spectroscopic properties of bones from a murine model of skeletal fragility (SAMP6). Intact bones from SAMP6 mice are weak and brittle compared with SAMR1 controls, a defect attributed to reduced strength of the bone matrix. The matrix weakness is attributed primarily to poorer organization of collagen fibers and reduced collagen content.

Aged mice have enhanced endocortical response and normal periosteal response compared with young-adult mice following 1 week of axial tibial compression.

With aging, the skeleton may lose its ability to respond to positive mechanical stimuli. We hypothesized that aged mice are less responsive to loading than young‐adult mice. We subjected aged (22 months) and young‐adult (7 months) BALB/c male mice to daily bouts of axial tibial compression for 1 week and evaluated cortical and trabecular responses using micro–computed tomography (µCT) and dynamic histomorphometry. The right legs of 95 mice were loaded for 60 rest‐inserted cycles per day to 8, 10, or 12 N peak force (generating mid‐diaphyseal strains of 900 to 1900 µε endocortically and 1400 to 3100 µε periosteally). At the mid‐diaphysis, mice from both age groups showed a strong anabolic response on the endocortex (Ec) and periosteum (Ps) [Ec.MS/BS and Ps.MS/BS: loaded (right) versus control (left), p < .05]. Generally, bone formation increased with increasing peak force. At the endocortical surface, contrary to our hypothesis, aged mice had a significantly greater response to loading than young‐adult mice (Ec.MS/BS and Ec.BFR/BS: 22 months versus 7 months, p < .001). Responses at the periosteal surface did not differ between age groups (p > .05). The loading‐induced increase in bone formation resulted in increased cortical area in both age groups (loaded versus control, p < .05). In contrast to the strong cortical response, loading only weakly stimulated trabecular bone formation. Serial (in vivo) µCT examinations at the proximal metaphysis revealed that loading caused a loss of trabecular bone in 7‐month‐old mice, whereas it appeared to prevent bone loss in 22‐month‐old mice. In summary, 1 week of daily tibial compression stimulated a robust endocortical and periosteal bone‐formation response at the mid‐diaphysis in both young‐adult and aged male BALB/c mice. We conclude that aging does not limit the short‐term anabolic response of cortical bone to mechanical stimulation in our animal model.

Connexin43 deficiency reduces the sensitivity of cortical bone to the effects of muscle paralysis

We have shown previously that the effect of mechanical loading on bone depends in part on connexin43 (Cx43). To determine whether Cx43 is also involved in the effect of mechanical unloading, we have used botulinum toxin A (BtxA) to induce reversible muscle paralysis in mice with a conditional deletion of the Cx43 gene in osteoblasts and osteocytes (cKO). BtxA injection in hind limb muscles of wild‐type (WT) mice resulted in significant muscle atrophy and rapid loss of trabecular bone. Bone loss reached a nadir of about 40% at 3 weeks after injection, followed by a slow recovery. A similar degree of trabecular bone loss was observed in cKO mice. By contrast, BtxA injection in WT mice significantly increased marrow area and endocortical osteoclast number and decreased cortical thickness and bone strength. These changes did not occur in cKO mice, whose marrow area is larger, osteoclast number higher, and cortical thickness and bone strength lower relative to WT mice in basal conditions. Changes in cortical structure occurring in WT mice had not recovered 19 weeks after BtxA injection despite correction of the early osteoclast activation and a modest increase in periosteal bone formation. Thus BtxA‐induced muscle paralysis leads to rapid loss of trabecular bone and to changes in structural and biomechanical properties of cortical bone, neither of which are fully reversed after 19 weeks. Osteoblast/osteocyte Cx43 is involved in the adaptive responses to skeletal unloading selectively in the cortical bone via modulation of osteoclastogenesis on the endocortical surface.

Biomechanical Evaluation of 2 Techniques for Ulnar Collateral Ligament Reconstruction of the Elbow

Background Elbow medial ulnar collateral ligament tears often result in pain and instability that may be career threatening in overhead-throwing athletes. Surgical reconstruction is frequently chosen to treat this injury. Ulnar collateral ligament reconstruction as described by Jobe is the most commonly used technique. Testing of this construct has not demonstrated that the biomechanical parameters of the native ligament are restored. A more recent construct, the docking technique, may more reliably reproduce these factors. Hypothesis Increasing the number of strands of palmaris longus tendon graft used in ulnar collateral ligament reconstruction and tensioning them using the docking technique result in a construct with improved biomechanical parameters as compared with the Jobe technique. Study Design Controlled laboratory study. Methods Thirty-three fresh-frozen human cadaveric elbows were randomized into 3 subgroups: Jobe (11), docking (12), and native (10). The Jobe and docking groups underwent reconstruction using their described palmaris tendon graft constructs. The ulnar collateral ligament was left intact in the native group. Elbows were potted and tested using a servohydraulic materials testing machine to apply a valgus moment at 30° of elbow flexion. Maximal moments to failure, stiffness, and strain at maximal moment and with a 3 N·m force applied were determined using a 2-camera motion analysis system to track reflective markers spanning the site. Results The docking (14.3 N·m) and native (18.8 N·m) subgroups resulted in higher maximal moment to failure than did the Jobe (8.9 N·m) subgroup (P <. 001). There was no significant difference between native and docking groups (P >. 05). Native ligaments were stiffer (301.4 N·m) than were Jobe (74.3 N·m) or docking (80.8 N·m; P <. 001). Native ligaments demonstrated lower strain at maximal force (0.087 mm/mm) and 3 N·m forces (0.030 mm/mm) than did the Jobe (0.198/0.057 mm/mm) or docking (0.287/0.042 mm/mm) subgroups. There was no difference in stiffness or strain between the Jobe and docking subgroups (P >. 05). Conclusion Neither technique reproduced the biomechanical profile of the native ulnar collateral ligament; the findings of this study suggest that the docking construct may offer initial biomechanical advantage over the Jobe construct.

Long Bones From the Senescence Accelerated Mouse SAMP6 Have Increased Size But Reduced Whole‐bone Strength and Resistance to Fracture

The senescence accelerated mouse strain P6 (SAMP6) has emerged as a useful model of senile osteoporosis because it has many features of the disease, including low trabecular bone formation and low areal bone density. We further characterized the SAMP6 model of senile osteoporosis by comparing morphological, mechanical, and densitometric properties of femurs and tibias from SAMP6 mice to those of the control strain (SAMR1) at 4 months and 12 months of age. SAMP6 long bones had increased periosteal width and endosteal area (p < 0.05), resulting in an average increase of 30% in moments of inertia (p < 0.05), but no difference in bone area (p > 0.05) compared with control. Despite their increased moments of inertia, long bones from SAMP6 mice were relatively weak and brittle. Ultimate bending moment was reduced by 25%, and both postyield displacement and energy‐to‐fracture were reduced by 60% compared with SAMR1 controls (p < 0.001). Average cortical ash fraction was increased slightly from 0.74 in SAMR1 to 0.76 in SAMP6 bones (p < 0.05), indicating that increased mineralization may have contributed to the brittleness of SAMP6 bones. The relative differences we observed—increased endosteal and periosteal dimensions, reduced bending strength, increased brittleness, and increased mineralization—are analogous to changes that occur in the aging human skeleton. Moreover, these features were consistently observed in young (4‐month) and old (12‐month) animals. These findings extend the previous descriptions of the SAMP6 mouse and identify key mechanical features that further validate its relevance as a unique and functionally relevant model of senile osteoporosis.