Ashlee E. Watts

Cell- and gene-based approaches to tendon regeneration

Repair of rotator cuff tears in experimental models has been significantly improved by the use of enhanced biologic approaches, including platelet-rich plasma, bone marrow aspirate, growth factor supplements, and cell- and gene-modified cell therapy. Despite added complexity, cell-based therapies form an important part of enhanced repair, and combinations of carrier vehicles, growth factors, and implanted cells provide the best opportunity for robust repair. Bone marrow-derived mesenchymal stem cells provide a stimulus for repair in flexor tendons, but application in rotator cuff repair has not shown universally positive results. The use of scaffolds such as platelet-rich plasma, fibrin, and synthetic vehicles and the use of gene priming for stem cell differentiation and local anabolic and anti-inflammatory impact have both provided essential components for enhanced tendon and tendon-to-bone repair in rotator cuff disruption. Application of these research techniques in human rotator cuff injury has generally been limited to autologous platelet-rich plasma, bone marrow concentrate, or bone marrow aspirates combined with scaffold materials. Cultured mesenchymal progenitor therapy and gene-enhanced function have not yet reached clinical trials in humans. Research in several animal species indicates that the concept of gene-primed stem cells, particularly embryonic stem cells, combined with effective culture conditions, transduction with long-term integrating vectors carrying anabolic growth factors, and development of cells conditioned by use of RNA interference gene therapy to resist matrix metalloproteinase degradation, may constitute potential advances in rotator cuff repair. This review summarizes cell- and gene-enhanced cell research for tendon repair and provides future directions for rotator cuff repair using biologic composites.

Gene therapy in musculoskeletal repair.

Abstract:  Local and regional gene therapy has improved healing in preclinical trials of articular and other muculoskeletal conditions. Combinations of cell supplementation and cells overexpressing growth factor genes have shown promising results for improving cartilage repair, enhancing delayed union of fractures, and driving organized tendon repair. Proof of concept has been developed using viral vectors, predominantly adenovirus, to deliver growth factor genes, such as BMP‐2, TGF‐β1, and IGF‐I. Integrating vectors, such as retrovirus and lentivirus, have improved the duration of gene‐induced repair, however, increased risk factors have limited broad application. Cartilage repair can be improved using chondrocyte or stem cell transplantation with cells expressing IGF‐I, BMP‐2, or FGF‐2. In cartilage injury and secondary osteoarthritis models, a combination of IL‐1 knockdown and growth factor supplementation has restored cartilage matrix and stabilized the osteoarthritic process. Ultimately, nonviral vectors may provide similar control of catabolic activity in cartilage and synovial structures, which may further improve outcome after chondrocyte or mesenchymal stem cell (MSC) implantation. MSCs derived from bone marrow, fat, or other connective tissues provide a multipotent cell source that may be privileged vectors for skeletal gene therapy. MSCs expressing BMP‐2, TGF‐β1, LMP‐1, IGF‐I, or GDF‐5 have enhanced cartilage, bone, and tendon repair. Overall, the field of orthopedic gene therapy for enhanced tissue repair has made significant preclinical advances. Combining existing cell transplant technology to deliver differentiated cells in a minimally invasive way, with genes that improve matrix formation, provides a manageable protocol for a persisting anabolic impact.

A collagenase gel/physical defect model for controlled induction of superficial digital flexor tendonitis

REASONS FOR PERFORMING STUDY A consistent and clinically relevant model for the induction of core lesions confined to the mid-metacarpal superficial digital flexor tendon (SDFT) has not been previously reported. Injection of bacterial collagenase is commonly used but often results in large, irregular and inconsistent lesions that disrupt the superficial tendon layers and epitenon. OBJECTIVE To develop and evaluate a new injection technique for collagenase induction of SDFT injury. METHODS Collagenase gel was injected into a physical columnar defect created by longitudinally placing a curved 16 gauge 8.89 cm needle in the mid-metacarpal SDFT in a randomly selected forelimb of 10 horses. A placebo treatment injection was performed 1 week later. Serial ultrasound examinations were performed. Horses were subjected to euthanasia at 2 (n = 2), 4 (n = 2), 8 (n = 4) and 16 (n = 2) weeks post treatment injection. Post mortem magnetic resonance imaging and histological analysis were performed. Gene expression (18S, SCX, TNC, TNMD, COL1A1, COL3A1, COMP, DCN, MMP1, MMP3 and MMP13), total DNA, glycosaminoglycan and collagen content were determined for experimental tendons (n = 10) and unaffected tendons (n = 9). RESULTS Mid-metacarpal SDFT core lesion induction was successful in all tendons with consistent lesion cross-sectional area and minimal epitenon disruption. Histology confirmed loss of normal tendon architecture after tendonitis induction and subsequent healing of the tendon core lesion. Compared with gene expression in unaffected tendons, several tested genes were significantly upregulated (COL1A1, COL3A1, TNMD, SCX, TNC, MMP13), while others showed significant downregulation (COMP, DCN, and MMP3). CONCLUSION Compared with the previously used direct injection of collagenase, this injection technique was easily performed and induced more consistent lesions that were mid-metacarpal and did not disrupt the epitenon. POTENTIAL RELEVANCE This model will allow for objective assessment of therapies for tendon regeneration in the mid-metacarpal SDFT prior to clinical trials and routine clinical application.

Accumulation of amikacin in synovial fluid after regional limb perfusion of amikacin sulfate alone and in combination with ticarcillin/clavulanate in horses.

OBJECTIVES To determine the effect of regional limb perfusion (RLP) with amikacin sulfate alone and in combination with ticarcillin/clavulanate on synovial fluid concentration and antimicrobial activity of amikacin. SAMPLE POPULATION Experimental study. METHODS RLP with amikacin alone (A; 2.5 g) or amikacin and ticarcillin/clavulanate (AT; 2.5 g amikacin, 7 g ticarcillin/clavulanate) was performed with a tourniquet placed at mid-antebrachium in standing, sedated horses. Perfusate blood was collected immediately after injection and again before tourniquet release. Blood from the jugular vein was collected before tourniquet release. Synovial fluid from the middle carpal joint was collected 0, 30, and 60 minutes after tourniquet release. Amikacin concentration and antimicrobial activity of synovial fluid against Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, and Pseudomonas aeruginosa were determined. RESULTS There was significantly lower amikacin concentration in the middle carpal joint synovial fluid of group AT compared with group A at 30 minutes (AT = median 4.4 µg/mL, IQR 3.0-11.2 µg/mL; A = 17.5 µg/mL, 6.6-80.1 µg/mL) and 60 minutes (AT = median 4.6 µg/mL, IQR 3.1-8.1 µg/mL; A = 15.0 µg/mL, 6.7-61.7 µg/mL) after tourniquet release. Zones of inhibition for ticarcillin-resistant Klebsiella pneumoniae from group AT were significantly smaller than group A from synovial fluid at 30 and 60 minutes after tourniquet release and in the perfusate serum before tourniquet release. CONCLUSIONS The combination of amikacin with ticarcillin/clavulanate during RLP resulted in significantly lower amikacin synovial concentration and antimicrobial activity on amikacin susceptible and ticarcillin resistant cultures compared with amikacin alone.

Effects of clopidogrel on the platelet activation response in horses

OBJECTIVE To evaluate the platelet activation response before and after treatment with clopidogrel in horses. ANIMALS 12 healthy adult mares. PROCEDURES In a masked study, horses (6/group) were randomly allocated to alternately receive placebo or clopidogrel via nasogastric tube at a loading dose of 4 mg/kg followed by 2 mg/kg every 24 hours. Blood samples were collected before and 72 hours after initiation of treatment for ADP- and collagen-induced light transmission aggregometry; determination of closure time in collagen-ADP cartridges; modified thrombelastography for comparison of maximal amplitudes generated by kaolin, reptilase, and reptilase plus ADP activation; and flow cytometric tests to detect platelet fibrinogen binding, P-selectin expression, and phosphatidylserine externalization before and after ex vivo stimulation with thrombin, convulxin, thrombin with convulxin, and calcium ionophore. RESULTS Clopidogrel administration induced a significant decrease in mean aggregation response to 5 μM and 10 μM ADP stimulation; however, 2 horses had resistance to clopidogrels inhibitory action. Significant differences after clopidogrel treatment were not found in any other tests of platelet function. CONCLUSIONS AND CLINICAL RELEVANCE Assays using commercially available reagents were configured to measure different variables of the platelet activation response; however, clopidogrels platelet inhibitory action was only detected by ADP-induced light transmission aggregometry. Results also suggested that horses, like humans, have interindividual variability in response to clopidogrel that may influence the drugs clinical efficacy as an antiplatelet agent.

Biopsy Needle Advancement during Bone Marrow Aspiration Increases Mesenchymal Stem Cell Concentration

Point-of-care kits to concentrate bone marrow (BM)-derived mesenchymal stem cells (MSCs) are used clinically in horses. A maximal number of MSCs per milliliter of marrow aspirated might be desired prior to use of a point-of-care system to concentrate MSCs. Our objective was to test a method to increase the number of MSCs per milliliter of marrow collected. We collected two BM aspirates using two different collection techniques from 12 horses. The first collection technique was to aspirate BM from a single site without advancement of the biopsy needle. The second collection technique was to aspirate marrow from multiple sites within the same sternal puncture by advancing the needle 5 mm three times for BM aspiration from four sites. Numbers of MSCs in collected BM were assessed by total nucleated cell count of BM after aspiration, total colony-forming unit-fibroblast (CFU-F) assay, and total MSC number at each culture passage. The BM aspiration technique of four needle advancements during BM aspiration resulted in higher initial nucleated cell counts, more CFU-Fs, and more MSCs at the first passage. There were no differences in the number of MSCs at later passages. Multiple advancements of the BM needle during BM aspiration resulted in increased MSC concentration at the time of BM collection. If a point-of-care kit is used to concentrate MSCs, multiple advancements may result in higher MSC numbers in the BM concentrate after preparation by the point-of-care kit. For culture expanded MSCs beyond the first cell passage, the difference is of questionable clinical relevance.

In Vitro Elution of Amikacin and Ticarcillin from a Resorbable, Self-Setting, Fiber Reinforced Calcium Phosphate Cement

OBJECTIVE To determine in vitro elution characteristics of amikacin and ticarcillin from fiber reinforced calcium phosphate beads (FRCP). SAMPLE POPULATION Experimental. METHODS FRCP beads with water (A), amikacin (B), ticarcillin/clavulanate (C), or both amikacin and ticarcillin/clavulanate (D) were bathed in mL phosphate-buffered saline (PBS) at 37°C, 5% CO(2) and 95% room air. PBS was sampled (eluent) and beads were placed in fresh PBS at time points 1 and 8 hours and 1, 2, 3, 4, 5, 6, 7, 10, 12, 14, 18, 21, 25, 28, 35, 42, 49, and 56 days. Antibiotic concentration and antimicrobial activity of eluent against Escherichia coli, Staphylococcus aureus, and Klebsiella pneumoniae were determined. RESULTS Both antibiotics eluted in a bimodal pattern. Beads with a single antibiotic eluted 20.8 ± 2.5% of amikacin and 29.5 ± 0.8% of ticarcillin over 56 days. Coelution of the antibiotics resulted in a lower proportion (AUC(0-∞) ) of antibiotics eluted for both amikacin (9.5 ± 0.2%) and ticarcillin (21.7 ± 0.09%). Bioassay of antimicrobial activity of the eluent (t = 1, 8, and 24 hours) established reduced antimicrobial activity of amikacin from combination beads (D). CONCLUSIONS FRCP beads with amikacin or ticarcillin/clavulanate, but not the combination, are suitable carriers for wound implantation. CLINICAL RELEVANCE Duration before complete resorption of FRCP beads in vivo should be determined before clinical use as a resorbable depot. The results of this study underscore the importance of testing drug combinations, despite success of the combination systemically, before their use in local applications.

Continuous peripheral neural blockade to alleviate signs of experimentally induced severe forelimb pain in horses

OBJECTIVE To investigate the efficacy and safety of a low-volume, single-catheter, continuous peripheral neural blockade (CPNB) technique to locally deliver bupivacaine to alleviate signs of severe forelimb pain resulting from experimentally induced tendonitis in horses. DESIGN Randomized controlled experimental trial. SAMPLE 14 horses and 5 forelimbs from equine cadavers. PROCEDURES Horses underwent collagenase-induced superficial digital flexor tendonitis in the midmetacarpal region of 1 forelimb. To deliver analgesia, a closed-tip catheter was placed from lateral to medial, approximately 12 cm distal to the accessory carpal bone, between the suspensory ligament and accessory ligament of the deep digital flexor tendon. Success of catheter placement and anesthetic delivery was documented ex vivo in 5 forelimbs from equine cadavers. Effective analgesia in affected forelimbs of horses from continuous (n = 7) versus intermittent (7) local anesthetic delivery (intermittent peripheral neural blockade; IPNB) was compared over a 3-day period. RESULTS Horses that received CPNB in the affected forelimb were less lame than horses that received IPNB. A lower proportion of CPNB-treated horses had behavioral and physiologic signs of pain, compared with IPNB-treated horses. Neither technique completely blocked the sensation of pain or resulted in swelling in the distal portion of the forelimb, vasodilation, or an increase in lameness. After removal, Staphylococcus aureus was cultured from 1 catheter tip. CONCLUSIONS AND CLINICAL RELEVANCE For short-term treatment, CPNB was more effective than IPNB for reduction in signs of severe pain in the distal aspect of the forelimb of horses.

Comparison of plasma and peritoneal indices of fibrinolysis between foals and adult horses with and without colic

OBJECTIVE To identify hemostatic imbalances indicative of an increased risk of intra-abdominal adhesion formation in foals versus adult horses. ANIMALS Horses with colic undergoing exploratory laparotomy or abdominocentesis as part of a clinical examination (n = 16 foals ≤ 6 months of age and 19 adults ≥ 5 years of age) and horses without colic undergoing herniorrhaphy (15 foals) or euthanasia for noninflammatory and nongastrointestinal disease (10 foals and 20 adults). PROCEDURES Paired abdominal fluid and blood samples were collected from each horse into buffered sodium citrate and centrifuged immediately after collection. Supernatants were stored at -80°C, then thawed for measurement of fibrinogen concentration, plasminogen activity, antiplasmin activity, and D-dimer concentration. Supernatant analyte concentrations or activities were compared within age group (foals with and without colic vs adults with and without colic) and within disease status (foals and adults without colic vs foals and adults with colic). RESULTS All analyte concentrations or activities in abdominal fluid samples were significantly lower in the noncolic groups than in the colic groups, and none differed between foal and adult groups. Several plasma analyte values differed by disease status and age. CONCLUSIONS AND CLINICAL RELEVANCE The risk of intra-abdominal adhesion formation in the foals in this study did not appear to be attributable to differences in intra-abdominal hemostasis between adult horses and foals. Strategies for initial medical and surgical management of colic in adult horses may be applicable to foals with similar disorders.

Effects of clopidogrel on horses with experimentally induced endotoxemia

OBJECTIVE To evaluate the effects of clopidogrel on clinical and clinicopathologic variables in healthy horses with experimentally induced endotoxemia. ANIMALS 12 adult mares. Procedures-Horses were assigned with a randomization procedure to receive clopidogrel (4 mg/kg, once, then 2 mg/kg, q 24 h; n = 6) or a placebo (6) through a nasogastric tube. After 72 hours of treatment, horses received lipopolysaccharide (LPS; 30 ng/kg, IV). Heart rate, respiratory rate, rectal temperature, CBC variables, plasma fibrinogen concentration, serum tumor necrosis factor-α concentration, plasma von Willebrand factor concentration, and measures of platelet activation (including ADP- and collagen-induced platelet aggregation and closure times, thrombelastography variables, and results of flow cytometric detection of platelet membrane P-selectin, phosphatidylserine, and microparticles) were determined at various times before and after LPS administration by investigators unaware of the treatment groups. Statistical analyses were performed with repeated-measures ANOVA. RESULTS 4 of 6 clopidogrel-treated horses had significant decreases in ADP-induced platelet aggregation before and after LPS administration. Heart rate increased significantly after LPS administration only for the placebo group. No significant differences were detected between groups for CBC variables, closure time, and plasma concentration of fibrinogen or serum concentration of tumor necrosis factor-α, and no clinically relevant differences were detected for other hemostatic variables. CONCLUSIONS AND CLINICAL RELEVANCE In this study, administration of LPS did not induce platelet hyperreactivity in horses on the basis of measures of platelet adhesion, aggregation, degranulation, and procoagulant activity. Administration of clopidogrel was associated with variable platelet antiaggregatory activity and attenuated some clinical signs of endotoxemia.