John J. Callaci

The Effect of Corticosteroid on Collagen Expression in Injured Rotator Cuff Tendon

BACKGROUND Subacromial corticosteroid injections are commonly used in the nonoperative management of rotator cuff disease. The effects of corticosteroid injection on injured rotator cuff tendons have not been studied. Our aims were to characterize the acute response of rotator cuff tendons to injury through the analysis of the type-III to type-I collagen expression ratio, a tendon injury marker, and to examine the effects of corticosteroid on this response. METHODS Sixty Sprague-Dawley rats were randomly assigned to four groups: control, tendon injury, steroid treatment, and tendon injury and steroid treatment. Six rats served as sham controls. Unilateral tendon injuries were created with full-thickness defects across 50% of the total width of the infraspinatus tendon, 5 mm from its humeral insertion. Steroid treatment with a single dose of methylprednisolone (0.6 mg/kg), equivalent to that given to humans, was injected into the subacromial space under direct visualization. Steroid treatment followed the creation of an injury in the rats in the injury and steroid treatment group. At one, three, and five weeks after the injury, the total RNA isolated from tendons was quantified with real-time polymerase chain reaction with use of primers for type-I and type-III collagen and ribosomal 18s RNA. RESULTS The type-III to type-I collagen expression ratio remained at baseline at all time-points in the control and sham groups. At one week, the type-III to type-I collagen expression ratio increased more than fourfold above the control level in the tendon injury group (p = 0.017) and the tendon injury and steroid treatment group (p = 0.003). The ratio remained greater than twofold above the control at three weeks in both groups (p = 0.003 and p = 0.037) and returned to baseline at five weeks. Interestingly, the group that had steroid treatment only showed an increase of >4.5-fold (p = 0.001) in the type-III to type-I collagen expression ratio, without structural injury to the tendon. This ratio returned to baseline levels by three weeks. CONCLUSIONS A single dose of corticosteroid does not alter the acute phase response of an injured rotator cuff tendon in the rat. However, the same steroid dose in uninjured tendons initiates a short-term response equivalent to that of structural injury.

Effect of corticosteroids on the biomechanical strength of rat rotator cuff tendon.

BACKGROUND The effect of corticosteroids on tendon properties is poorly understood, and current data are contradictory and diverse. The biomechanical effect of steroids on rotator cuff tendon has not been studied, to our knowledge. The current study was undertaken to characterize the biomechanical effects of corticosteroid exposure on both uninjured and injured rat rotator cuff tendon. METHODS One hundred and twenty-three male Sprague-Dawley rats were randomly assigned to four groups: control (C), tendon injury (I), steroid exposure (S), and tendon injury plus steroid exposure (I+S). Unilateral tendon injuries consisting of a full-thickness defect across 50% of the total width of the infraspinatus tendon were created. Steroid treatment consisted of a single dose of methylprednisolone placed into the subacromial space. At one, three, and five weeks postoperatively, the shoulders were harvested and the infraspinatus tendon was subjected to biomechanical testing. Two specimens from each group were used for histological analysis. RESULTS At one week, maximum load, maximum stress, and stiffness were all significantly decreased in Group S compared with the values in Group C. Mean maximum load decreased from 37.9 N in Group C to 27.5 N in Group S (p < 0.0005). Mean maximum stress decreased from 18.1 MPa in Group C to 13.6 MPa in Group S (p < 0.0005). Mean stiffness decreased from 26.3 N/mm in Group C to 17.8 N/mm in Group S (p < 0.0005). At one week, mean maximum stress in Group I+S (17.0 MPa) was significantly decreased compared with the value in Group I (19.5 MPa) (p < 0.0005). At both the three-week and the five-week time point, there were no significant differences between Group C and Group S or between Group I and Group I+S with regard to mean maximum load, maximum stress, or stiffness. Histological analysis showed fat cells and collagen attenuation in Groups S and I+S. These changes appeared to be transient. CONCLUSIONS A single dose of corticosteroids significantly weakens both intact and injured rat rotator cuff tendons at one week. This effect is transient as the biomechanical properties of the steroid-exposed groups returned to control levels by three weeks.

The Biomechanical and Histologic Effects of Platelet-Rich Plasma on Rat Rotator Cuff Repairs

Background: Rotator cuff tears are common injuries that are often treated with surgical repair. Because of the high concentration of growth factors within platelets, platelet-rich plasma (PRP) has the potential to enhance healing in rotator cuff repairs. Hypothesis: Platelet-rich plasma would alter the biomechanical and histologic properties of rotator cuff repair during an acute injury response. Study Design: Controlled laboratory study. Methods: Platelet-rich plasma was produced from inbred donor rats. A tendon-from-bone supraspinatus tear was created surgically and an immediate transosseous repair performed. The control group underwent repair only. The PRP group underwent a repair with PRP augmentation. Rats in each group were sacrificed at 7, 14, and 21 days. The surgically repaired tendons underwent biomechanical testing, including failure load, stiffness, failure strain, and stress relaxation characteristics. Histological analysis evaluated the cellular characteristics of the repair tissue. Results: At 7- and 21-day periods, augmentation with PRP showed statistically significant effects on the biomechanical properties of the repaired rat supraspinatus tear, but failure load was not increased at the 7-, 14-, or 21-day periods (P = .688, .209, and .477, respectively). The control group had significantly higher stiffness at 21 days (P = .006). The control group had higher failure strain at 7 days (P = .02), whereas the PRP group had higher failure strain at 21 days (P = .008). Histologically, the PRP group showed increased fibroblastic response and vascular proliferation at each time point. At 21 days, the collagen fibers in the PRP group were oriented in a more linear fashion toward the tendon footprint. Conclusion: In this controlled, rat model study, PRP altered the tissue properties of the supraspinatus tendon without affecting the construct’s failure load. Clinical Relevance: The decreased tendon tissue stiffness acutely and failure to enhance tendon-to-bone healing of repairs should be considered before augmenting rotator cuff repairs with PRP. Further studies will be necessary to determine the role of PRP in clinical practice.

Binge alcohol treatment of adolescent rats followed by alcohol abstinence is associated with site-specific differences in bone loss and incomplete recovery of bone mass and strength

We previously demonstrated that alcohol-fed adolescent rats exhibit reductions in lumbar spine bone mineral density (BMD) and vertebral body height, suggesting that chronic alcohol consumption has negative consequences for skeletal development during adolescence. Binge alcohol consumption is common in adolescents and young adults, yet little is known about its consequences on skeletal integrity or the attainment of peak bone mass. We used a previously validated binge alcohol exposure model to test the hypothesis that binge alcohol treatment of adolescent rats would be associated with distinct temporal and site-specific bone loss profiles, with incomplete recovery from bone loss following a period of alcohol abstinence. Seventy-two male adolescent Sprague-Dawley rats were assigned to one of six treatment groups (n=12/group) receiving binge alcohol (3 g/kg) or saline intraperitoneal, 3 consecutive days (acute binge), 4 consecutive weekly (3-day) binge cycles (chronic binge), or 4 weekly binge cycles followed by a 30-day abstinence period without alcohol or saline injections (chronic binge with abstinence). Cancellous BMD was determined by quantitative computed tomography and compressive strength determined by biomechanical testing. Serum testosterone and osteocalcin levels were measured by enzyme-linked immunosorbent assay. Tibial cancellous BMD was significantly reduced by 25% (P<.05) after both acute and chronic binge alcohol treatment and vertebral cancellous BMD was significantly reduced by 15% (P<.05) after chronic binge exposure. Vertebral compressive strength was also significantly decreased by 31% (P<.05) after chronic binge alcohol treatment. Tibial cancellous BMD returned to control levels after the 30-day alcohol abstinence period, but vertebral cancellous BMD remained 15% below control values (P<.05) 30 days after termination of binge alcohol exposures. Serum osteocalcin levels were significantly decreased following acute binge alcohol exposure (P<.05). These results show that binge alcohol exposure can produce both short- and long-term skeletal damage in the adolescent rat. These data might have relevance to peak bone mass attainment and future risk of skeletal disease in adolescents and young adults who engage in repeated binge-drinking episodes.

The Effects of Binge Alcohol Exposure on Bone Resorption and Biomechanical and Structural Properties are Offset by Concurrent Bisphosphonate Treatment

BACKGROUND Chronic alcohol consumption reduces bone mass and strength, increasing fracture risk for alcohol abusers. Mechanisms underlying this vulnerability involve modulation of bone remodeling. Direct effects of alcohol on bone formation have been documented; those on bone resorption are less well studied. Skeletal effects of exposure to high blood alcohol concentrations (BACs) attained during binge drinking have not been studied. We examined the effects of repeated binge-like alcohol treatment on bone resorption, bone mineral density and vertebral compressive strength in adult male rats treated with the aminobisphosphonate, risedronate. METHODS A binge alcohol exposure model was developed using intraperitoneal (IP) injection to administer a 20% (vol/vol) alcohol/saline solution (3 g/kg, 1X/day) on four consecutive days for 1, 2 or 3 weeks in 400 g rats, with and without weekly risedronate treatment (0.5 mg/kg, 1X/week). Total serum deoxypyridinoline (Dpd) a crosslink of bone type collagen released during resorption was measured by ELISA. Bone mineral density (BMD) was measured using peripheral quantitative computed tomography (pQCT). Vertebral compressive strength was determined using an Instron materials testing machine. Trabecular integrity was analyzed by computer-aided trabecular analysis system (TAS). RESULTS Peak BACs averaged 308.5 +/- 12 mg/dL; average BAC was 258.6 +/- 28.7 mg/dL at time of euthanasia. No significant effects of treatment were observed after 1 or 2 weeks of binge alcohol exposure. At 3 weeks of alcohol treatment serum Dpd was significantly increased (205%, p < 0.05) over controls. Bone mineral density (BMD) in cancellous bone of distal femur and lumbar spine were significantly decreased (34% and 21% respectively, p < 0.01) after 3 weeks of binge treatment. Vertebral (L4) compressive strength (maximum load sustained before failure) also decreased (27%, p < 0.05) after 3 binge alcohol cycles. Risedronate maintained the Dpd level (p < 0.01), BMD (p < 0.001) and vertebral structural biomechanical properties (p < 0.01) of binge-treated rats at control levels (E vs ER). Indices of trabecular architectural integrity [Trabecular bone volume/tissue volume (BV/TV), bone area (BAR) and trabecular separation (Tb.Sp)] analyzed at week 3 showed (BV/TV) and (BAR) were significantly reduced in alcohol-binged rats (p < 0.01), while (Tb.Sp) was significantly increased (p < 0.01). Risedronate also maintained the trabecular architectural indices of binge-treated rats at control levels (E versus ER, p < 0.01). CONCLUSIONS In adult male rats, BACs reflective of those attained during alcoholic binge drinking may affect the skeleton in part by stimulating bone resorption, an effect mitigated by risedronate.

Distal tibiofibular bone-bridging in transtibial amputation.

BACKGROUND The creation of a bone bridge between the residual tibia and fibula is a controversial surgical technique used in the performance of transtibial amputation. METHODS Twenty consecutive patients who underwent a unilateral transtibial amputation, as a consequence of traumatic injury, had distal tibiofibular bone-bridging performed by a single surgeon. Eight completed the Prosthesis Evaluation Questionnaire (PEQ), a validated outcomes instrument designed to measure patient self-reported health-related quality of life after a lower-extremity amputation. Their responses were compared with those of a previously reported control group of nondiabetic patients who had undergone transtibial amputation with the use of a traditional technique and with those of a previously reported consecutive group of Brazilian patients, including twelve who were diabetic, who had undergone a similar bone-bridge procedure. RESULTS The scores in the American bone-bridge group were similar to those in the control group and not as good as those in the Brazilian bone-bridge group. The American bone-bridge and control groups scored lower in the Social Burden, Ambulation, Frustration, Sounds, Utility, and Well-Being domains of the PEQ. CONCLUSIONS While many experts in the care of amputees believe that the distal tibiofibular bone-bridge technique improves patient functional outcomes, our small group of patients treated with this procedure did not appear to have better outcomes than a group of patients treated successfully with a standard surgical technique. More information is needed before the bone-bridge technique can be recommended as an important component of standard transtibial amputation surgery.

Alcohol Exposure and Mechanisms of Tissue Injury and Repair

Tissue injury owing to acute and chronic alcohol consumption has extensive medical consequences, with the level and duration of alcohol exposure affecting both the magnitude of injury and the time frame to recovery. While the understanding of many of the molecular processes disrupted by alcohol has advanced, mechanisms of alcohol-induced tissue injury remain a subject of intensive research. Alcohol has multiple targets, as it affects diverse cellular and molecular processes. Some mechanisms of tissue damage as a result of alcohol may be common to many tissue types, while others are likely to be tissue specific. Here, we present a discussion of the alcohol-induced molecular and cellular disruptions associated with injury or recovery from injury in bone, muscle, skin, and gastric mucosa. In every case, the goal of characterizing the sites of alcohol action is to devise potential measures for protection, prevention, or therapeutic intervention.

REGULATION OF THE INDUCIBLE NITRATE REDUCTASE ISOFORM FROM SOYBEANS

The activity of the pH 7.5 NADH-linked nitrate reductase isoform from soybeans is termed inducible. Activity can be observed only in seedlings which have been supplied nitrate. Steady-state levels of mRNA for this isoform also show an absolute requirement for nitrate. Nitrate reductase specific mRNA can be observed within 2 h after nitrate treatment. Levels peaked 48 h after nitrate treatment, while the addition of glutamine to nitrate diminished amounts of nitrate reductase specific mRNA. Using nuclear runoff transcription assays, we have shown that one level of control of nitrate reductase synthesis is transcriptional.

Mesenchymal Stem Cells Facilitate Fracture Repair in an Alcohol-Induced Impaired Healing Model

Objectives: Clinical studies have shown alcohol to be a risk factor for traumatic orthopaedic injuries and for nonunion. Data from animal studies suggest that alcohol exposure inhibits fracture healing. This report presents a novel rodent model of impaired fracture healing caused by repeated alcohol exposure. Using this model, we examined the regenerative effects of an intravenously administered population of isolated and expanded mesenchymal stem cells (MSCs) on fracture healing. Methods: Bone marrow–derived MSC were isolated from transgenic green fluorescent protein C57BL/6 mice, and culture expanded using a lineage depletion protocol. Adult wild-type C57BL/6 mice were subjected to a 2-week binge alcohol exposure paradigm (3 days during which they received daily intraperitoneal injections of a 20% alcohol/saline solution followed by a 4-day rest period and another binge cycle for 3 consecutive days). At completion of the second binge cycle, mice were subjected to a mid-shaft tibia fracture while intoxicated. Twenty-four hours after the fracture, animals were administered an intravenous transplant of green fluorescent protein–labeled MSC. Two weeks after the fracture, animals were euthanized and injured tibiae were collected and subjected to biomechanical, histologic, and microcomputed tomography analysis. Results: Pre-injury binge alcohol exposure resulted in a significant impairment in biomechanical strength and decrease in callus volume. MSC transplants restored both fracture callus volume (P < 0.05) and biomechanical strength (P < 0.05) in animals with alcohol-impaired healing. In vivo imaging demonstrated a time-dependent MSC migration to the fracture site. Conclusions: These data suggest that a 2-week binge alcohol exposure significantly impairs fracture healing in a murine tibia fracture model. Intravenously administered MSC were capable of specifically homing to the fracture site and of normalizing biomechanical, histologic, and microcomputed tomography parameters of healing in animals exposed to alcohol. Understanding MSC recruitment patterns and functional contributions to fracture repair may lead to their use in patients with impaired fracture healing and nonunion.

Identification of Novel Bone-Specific Molecular Targets of Binge Alcohol and Ibandronate by Transcriptome Analysis

BACKGROUND Our laboratory established that binge alcohol-related bone damage is prevented by aminobisphosphonates, suggesting bone resorption increases following binge exposure. We examined the effects of binge alcohol and antiresorptive therapy on the relationship between bone damage and modulation of the vertebral transcriptome, in an attempt to determine how alcohol-induced bone damage and its prevention modulate bone-related biological pathways. METHODS Male Sprague-Dawley rats were assigned to 1 of 6 treatment groups (n = 12/group). (C1) saline ip 3 d/wk for 1 week, (A1) binge alcohol, 3 g/kg, ip 3 d/wk for 1 week, (C4) saline ip, 3 d/wk for 4 weeks, (A4) binge alcohol, ip, 3 g/kg 3 d/wk for 4 weeks, (I4) ibandronate, saline ip 3 d/wk for 4 weeks, plus a single ip injection of ibandronate at 120 microg/animal, and (AI4) binge alcohol plus ibandronate as above. After 1 or 4 weeks, adjacent lumbar vertebrae were assayed for bone damage or transcriptional changes. RESULTS Bone loss was not observed after 1 week of binge alcohol treatment. After 4 weeks, binge alcohol decreased vertebral BMD by 23% (p < 0.05) and compressive strength by 18% compared to saline controls (p < 0.05). Concurrent ibandronate prevented bone loss, increasing these parameters by 145 and 134% respectively compared to binge alcohol. (p < 0.05). Analysis of the vertebral transcriptome identified gene clusters specific for acute and chronic binge alcohol-related bone damage. Acute binge alcohol modulated the expression of integrin signaling-specific genes, while chronic binge alcohol modulated canonical Wnt signaling gene expression. Ibandronate normalized the expression of approximately 20% of the genes affected by chronic binge alcohol, allowing the identification of a unique subset of alcohol-sensitive, ibandronate-responsive genes. CONCLUSIONS Identification of bone-specific gene expression clusters associated with acute and chronic binge alcohol treatment allowed for the identification of cellular pathways affected by binge treatment with known involvement in bone remodeling (Integrin, Canonical Wnt signaling) not previously identified as alcohol-sensitive. This data provides a basis for a plausible mechanistic explanation for the known detrimental effects of alcohol on bone formation and resorption.