A.M. Pawde

Comparison of autogenic and allogenic bone marrow derived mesenchymal stem cells for repair of segmental bone defects in rabbits.

Autogenic and allogenic bone marrow derived mesenchymal stem cells (BM-MSCs) were compared for repair of bone gap defect in rabbits. BM-MSCs were isolated from bone marrow aspirates and cultured in vitro for allogenic and autogenic transplantation. A 5mm segmental defect was created in mid-diaphysis of the radius bone. The defect was filled with hydroxyapatite alone, hydroxyapatite with autogeneic BM-MSCs and hydroxyapatite with allogenic BM-MSCs in groups A, B and C, respectively. On an average 3.45×10(6) cells were implanted at each defect site. Complete bridging of bone gap with newly formed bone was faster in both treatment groups as compared to control group. Histologically, increased osteogenesis, early and better reorganization of cancellous bone and more bone marrow formation were discernible in treatment groups as compared to control group. It was concluded that in vitro culture expanded allogenic and autogenic BM-MSCs induce similar, but faster and better healing as compared to control.

Evaluation of autologous bone marrow‐derived nucleated cells for healing of full‐thickness skin wounds in rabbits

The aim of the study was to evaluate the potential of autologous bone marrow‐derived nucleated cells to enhance the rate of healing of full‐thickness excisional skin wounds in rabbits. The study was conducted on 20 New Zealand white rabbits of either sex. Two, 2 × 2 cm full‐thickness skin (thoracolumabar region) excisional wounds were created; one on each side of the dorsal midline in each animal. The wounds were randomly assigned to either injection of autologous bone marrow‐derived nucleated cells into the wound margins (BI), or topical application of sterile saline solution (normal saline, NS), which served as control. The wound healing was assessed by evaluation of granulation tissue formation, wound contraction, epithelisation and histopathological and histochemical changes up to 28 days after creation of the wound. Granulation tissue appeared significantly faster in BI‐treated wounds (3.22 ± 0.22 days) than in NS‐treated wounds (4.56 ± 0.47 days). Better epithelisation was seen histologically in BI wounds than in NS‐treated wounds. Wound contraction was significantly more in BI wounds when compared with NS wounds on 21 post‐surgery. Histopathological examination of the healing tissue showed early disappearance of inflammatory reaction, significantly more neovascularisation, and more fibroplasias and early lay down and histological maturation of collagen in BI wounds than in control wounds. It was concluded that injection of autologous bone marrow‐derived nucleated cells in the wound margins induced faster and better quality healing of excisional skin wounds in rabbits when compared with normal saline. The injection of autologous bone marrow‐derived nucleated cells can be used to promote healing of large full‐thickness skin wounds in rabbits.

Autologous bone marrow-derived cells for healing excisional dermal wounds of rabbits

The wound-healing potential of autologous bonemarrow-derived nucleated cells was evaluated infull-thickness skin wounds in the thoracolumbarregion of 20 clinically healthy rabbits. Three woundsof 2 x 2 cm, one on the left side and two right of themidline, were created on the dorsal lumbar regionof each rabbit under xylazine-ketamine anaesthesia.The wounds of each animal were randomly assignedto one of three treatments: injection of autologousbone marrow-derived cells into wound margins (BI),topical application of bone marrow-derived cellsover the wound surface (BT) or 5 per cent povidoneiodine solution (PI) (control). Wounds were observedfor 28 days for granulation tissue formation, woundcontraction, histomorphological and histochemicalevaluation, and time to complete healing. The mean(se) time to appearance of granulation tissue wassignificantly less in BI-treated wounds (3·22 [0·22] days)than the BT-treated (3·89 [0·40] days) and PI-treated(4·89 [0·47] days) groups. On days 14 and 21 aftersurgery, wound contraction was significantly (P<0·05)higher in BI-treated wounds (73·00 and 97·35 per cent)than in those treated with BT (58·75 and 84·87 per cent)and PI (54·84 and 84·60 per cent). Histomorphologicalfindings showed an earlier disappearance ofinflammatory reaction, better epithelialisation,significantly more neovascularisation, more fibroplasiaand collagenation, and earlier histological maturation inBI- and BT-treated wounds than in control wounds.

Management of tibial fractures using a circular external fixator in two calves.

OBJECTIVES To report the repair of tibial diaphyseal fractures in 2 calves using a circular external skeletal fixator (CEF). STUDY DESIGN Clinical report. ANIMALS Crossbred calves (n=2; age: 6 months; weight: 55 and 60 kg). METHODS Mid-diaphyseal tibial fractures were repaired by the use of a 4-ring CEF (made of aluminum rings with 2 mm K-wires) alone in 1 calf and in combination with hemicerclage wiring in 1 calf. RESULTS Both calves had good weight bearing with moderate lameness postoperatively. Fracture healing occurred by day 60 in 1 calf and by day 30 in calf 2. The CEF was well maintained and tolerated by both calves through fracture healing. Joint mobility and limb usage improved gradually after CEF removal. CONCLUSIONS CEF provided a stable fixation of tibial fractures and healing within 60 days and functional recovery within 90 days. CLINICAL RELEVANCE CEF can be safely and successfully used for the management of selected tibial fractures in calves.

Management of fractures near the carpal joint of two calves by transarticular fixation with a circular external fixator.

A four-ring circular external skeletal fixation device was evaluated for transcarpal fixation of compound fractures in two calves. Case 1 was an eight-month-old female Holstein-Friesian × indigenous breed calf weighing 72 kg, which had a Salter-Harris type II fracture at the distal metaphysis of the right radius/ulna with an open contaminated wound on the medial aspect of the carpus. Case 2 was an 18-month-old crossbred Haryana heifer weighing 105 kg, which had a comminuted fracture at the proximal end of its left metacarpus with severe soft tissue trauma and an open wound on the medial aspect. In both cases, the fractures were repaired with four-ring circular fixators by fixing the proximal two rings in the distal radius/ulna and the distal two rings in the metacarpus. Postoperatively, both calves were treated with analgesic and anti-inflammatory drugs and antibiotic, and the pin-bone interfaces and the open wound were cleaned regularly with povidone-iodine solution. In both animals weight bearing was good to excellent in the immediate postoperative period. The fixator was well maintained and tolerated by the animals until the fractures healed after 45 to 60 days. The movement of the carpal joint was slightly affected when the fixator was removed on day 60, but a follow-up examination after one year showed that both calves had normal functional usage of the limbs.

Effect of IGF-1 and Uncultured Autologous Bone-Marrow-Derived Mononuclear Cells on Repair of Osteochondral Defect in Rabbits

Objective: To investigate the utility of bone-marrow-derived mononuclear cells (BMNCs) and insulin like growth factor-1 (IGF-1) in articular cartilage repair. Design: An osteochondral defect of 3 mm diameter and 5 mm depth was created in patellar groove of the left knee joint in each of 36 New Zealand White rabbits. The defect was filled with RPMI-1640 medium in group A (control), autologous BMNCs in group B, and autologous BMNCs plus IGF-1 in group C (n = 12). Healing of the defect was assessed by gross, scanning electron microscopic, radiographic, and histological examinations up to 90 days. Results: Gross and scanning electron microscopic examination of the healing site revealed superior gross morphology and surface architecture of the healing tissue in the animals of group C as compared to other groups. Radiographically on day 90, the defect area was not distinguishable from the surrounding area in group C, but a small circular defect area was still evident in groups A and B. The regenerated tissue was mostly hyaline in group C and fibrocartilage in groups A and B. The cells were well organized and showed better deposition of proteoglycans in groups C and B than in group A. Conclusions: It was concluded that implantation of bone-marrow-derived nucleated cells may facilitate the healing of osteochondral defects; however, the combination of BMNCs and IGF-1 induces faster and histologically better healing than the BMNCs alone.

Effects of Epidural Ketamine–Xylazine Combination on the Clinicophysiological and Haematobiochemical Parameters of Uraemic and Healthy Goats

Xylazine–ketamine combination was evaluated for its efficacy and safety after epidural administration in uraemic and healthy goats. The combination (xylazine 0.025 mg/kg and ketamine 2.5 mg/kg) was administered to uraemic (n = 6) and healthy (n = 6) animals in the lumbosacral epidural space. The combination was evaluated in terms of clinical, physiological, haematological and biochemical parameters. The onset of analgesia was faster in healthy animals than in uraemic animals. Xylazine and ketamine produced complete analgesia of tail, perineum, inguinal and thigh regions in all animals of both groups. However, healthy animals showed longer duration of complete analgesia than did uraemic animals. Greater ataxia was recorded in healthy animals than in uraemic animals. The heart rate showed a significant decrease in both groups; however, respiratory rate and rectal temperature did not show any significant changes. Haemoglobin, packed cell volume and total leukocyte count decreased non-significantly in both groups. Total leukocyte count was significantly higher in uraemic animals. A significantly higher value of urea nitrogen and creatinine was recorded in uraemic animals. The blood electrolytes (Na+, K+ and Cl−) and blood gases (Po2 and Pco2) did not show any significant changes in both groups; however, base excess was significantly higher in uraemic animals. The effects produced by the combination on different systems were transient and values normal as the effect of the drugs wore off. The results suggest that the combination when used epidurally in uraemic goats produced effective and safe surgical analgesia.

Development of loop-mediated isothermal amplification (LAMP) for detection of Babesia gibsoni infection in dogs

Diagnosis of canine babesiosis, caused by Babesia gibsoni is difficult, especially in chronically infected dogs. A loop mediated isothermal amplification (LAMP) assay was developed and standardized by using four oligonucleotide primers targeting the hypervariable region of 18S rRNA gene (GenBank Acc. no. KC461261). The primers specifically amplified B. gibsoni DNA, while no amplification was detected with DNA from non-infected dogs as well as from dogs infected with Babesia canis vogeli, Hepatozoon canis, Ehrlichia canis and Trypanosoma evansi. The assay could detect 1.35 × 10(-7) parasitaemia and 10(-4) dilution of recombinant plasmid, equivalent to 12 pg of target DNA. All the samples were tested by nested PCR as well as LAMP assay. LAMP was found to be 10 times more sensitive than nested PCR targeting the same gene. Out of 75 suspected field samples, collected from different parts of the country, LAMP could detect B. gibsoni in 43 samples, while nested PCR and microscopy could detect 37 and 23 samples, respectively. High sensitivity, specificity and rapidity of LAMP assay may be exploited for screening large number of samples in a field setting.

Comparative Evaluation of In Vitro Mechanical Properties of Different Designs of Epoxy-Pin External Skeletal Fixation Systems

OBJECTIVES To compare in vitro biomechanical properties of different designs of epoxy-pin external skeletal fixator (ESF) constructs. STUDY DESIGN Mechanical testing study. SAMPLE POPULATION Four epoxy-pin ESF design constructs (uniplanar [EU], multiplanar-I [EM-I], multiplanar-II [EM-II], and circular [EC]) were mechanically tested in compression, bending, and torsion. METHODS Four different designs of free-form epoxy-pin external fixator constructs were developed using 1.5 mm K-wires and epoxy resin mounted in an ultra-high density polyethylene rod (20 mm diameter). Three-point fixation was done in each fragment, and the distance between fixation wires, and between the rod and the side bars was kept constant in all the designs. A 5 mm gap was maintained at the center of the fixation rod to simulate an unstable fracture condition. The fixator constructs (n = 12 of each design) were subjected to mechanical testing in axial compression, bending, or torsion. Load-deformation curves were generated and mechanical properties were compared between construct types. RESULTS EU was the weakest design. Under compression, constructs EM-I, EM-II, and EC were similar. Under bending, EM-I and EM-II had similar strength, whereas EC was strongest. Under torsion, EC was strongest, followed by EM-II, EM-I, and EU; EM-II provided double the rotational stability of EM-I. CONCLUSIONS Overall, EC followed by EM-II epoxy-pin fixator designs had better mechanical strength.

Mesenchymal stem cells with IGF-1 and TGF- β1 in laminin gel for osteochondral defects in rabbits

OBJECTIVE Healing of articular cartilage is still a challenge due to its limited potential to regenerate. In the present study, we evaluated allogenic bone marrow mesenchymal stem cells (BM-MSCs) alone or in combination with growth factors, insulin-like growth factor-1 (IGF-1) and transforming growth factor-β1 (TGF-β1) in laminin scaffolds for healing of osteochondral defects. DESIGN Osteochondral defects of 4mm (diameter) x 5mm (depth) were induced in the rabbit knee joints and treated with phosphate-buffered saline (PBS; control), BM-MSCs, BM-MSCs in laminin, BM-MSCs in laminin with IGF-1, or BM-MSCs in laminin with IGF-1 and TGF-β1 in 10 animals each. Gross, radiographic, scanning electron microscopic (SEM) and histologic examinations besides chondrocyte-specific genes expression by quantitative real time qPCR were carried out at 8 and 12 weeks. RESULTS Gross and SEM examination revealed superior morphology and surface architecture of the healing site in animals that received MSCs with IGF-1 or IGF-1 and TGF-β1. The application of laminin composites containing MSCs with IGF-1 and TGF-β1 significantly enhanced hyaline cartilage formation with improved cellular arrangement, proteoglycan deposition, clear tidemark zone and subchondral bone formation. However, regenerated tissue in defects that received only MSCs had poor tidemark zone and proteoglycans deposition Aggrecan and Coll2 expression was significantly higher in case of MSCs with growth factors. CONCLUSION The treatment with BM-MSCs combined with IGF-1/TGF-β1 into laminin gel scaffold might enhance the restoration of hyaline cartilage in osteochondral defect.