Equine adipose-tissue derived mesenchymal stem cells and platelet concentrates: their association in vitro and in vivo

Equine mesenchymal stem cells (MSC) are of particular interest both for basic research and for the therapeutic approach to musculoskeletal diseases in the horse. Their multilineage differentiation potential gives them the capability to contribute to the repair of tendon, ligament and bone damage. MSCs are also considered a promising therapeutic aid in allogeneic cell transplantation, since they show low immunogenicity and immunomodulating functions.Adipose tissue-derived adult equine stem cells (AdMSC) can be isolated, expanded in vitro and then inoculated into the damaged tissue, eventually in the presence of a biological scaffold. Here we report our preliminary experience with adipose-derived mesenchymal stem cells in allogeneic cell-therapy of tendonitis in the horse. MSCs, derived from visceral adipose tissue, were grown in the presence of autologous platelet lysate and characterized for their differentiation and growth potential. Expanded AdMSC were inoculated into the damaged tendon after their dispersion in activated platelet-rich plasma (PRP), a biological scaffold that plays an important role in maintaining cells in defect sites and contributes to tissue healing. Fourteen out of sixteen treated horses showed a functional recovery and were able to return to their normal activity.     

The effect of intralesional injection of bone marrow derived mesenchymal stem cells and bone marrow supernatant on collagen fibril size in a surgical model of equine superficial digital flexor tendonitis

Reasons for performing study: Collagen fibril size is decreased in repair tissue following tendon injury compared to normal tendon matrix in horses. Mesenchymal stem cells have been suggested to promote regeneration of tendon matrix rather than fibrotic repair following injury, although this concept remains unproven. Objectives: To explore the hypothesis that implantation of autologous mesenchymal stem cells derived from bone marrow into a surgically created central core defect in the superficial digital flexor tendon (SDFT) of horses would induce the formation of a matrix with greater ultrastructural similarities to tendon matrix than the fibrotic scar tissue formed in control defects. Methods: Tissue was collected 16 weeks after induction of injury and 12 weeks after treatment from normal and injured regions of control and treated limbs of 6 horses and examined using transmission electron microscopy. Collagen fibril diameters were measured manually with image analysis software and surface areas calculated. Three parameters assessed for normal and injured tissue were mass average diameter (MAD), collagen fibril index (CFI) and the area dependent diameter (ADD). Results: Normal regions from both treated and control limbs displayed higher MAD and CFI values, as well as a characteristic bimodal distribution in fibril size. Injured regions from both treated and control limbs displayed significantly lower MAD and CFI values, as well as a unimodal distribution in fibril size. There were no significant differences between treated and control limbs for any of the parameters assessed. Conclusions: Intralesional injection of autologous bone marrow derived mesenchymal stem cells had no measurable effect on the fibril diameter of collagen in healing tissue in the SDFT of this experimental model 16 weeks after injury. Potential relevance: Favouring matrix regeneration over fibrotic repair may not be the mechanism by which autologous mesenchymal stem cells assist healing of tendon injury.     

Equine adipose-tissue derived mesenchymal stem cells and platelet concentrates: their association in vitro and in vivo

Equine mesenchymal stem cells (MSC) are of particular interest both for basic research and for the therapeutic approach to musculoskeletal diseases in the horse. Their multilineage differentiation potential gives them the capability to contribute to the repair of tendon, ligament and bone damage. MSCs are also considered a promising therapeutic aid in allogeneic cell transplantation, since they show low immunogenicity and immunomodulating functions.Adipose tissue-derived adult equine stem cells (AdMSC) can be isolated, expanded in vitro and then inoculated into the damaged tissue, eventually in the presence of a biological scaffold. Here we report our preliminary experience with adipose-derived mesenchymal stem cells in allogeneic cell-therapy of tendonitis in the horse. MSCs, derived from visceral adipose tissue, were grown in the presence of autologous platelet lysate and characterized for their differentiation and growth potential. Expanded AdMSC were inoculated into the damaged tendon after their dispersion in activated platelet-rich plasma (PRP), a biological scaffold that plays an important role in maintaining cells in defect sites and contributes to tissue healing. Fourteen out of sixteen treated horses showed a functional recovery and were able to return to their normal activity.     

Monitoring the fate of autologous and allogeneic mesenchymal progenitor cells injected into the superficial digital flexor tendon of horses: preliminary study

Autologous mesenchymal progenitor cells (MPCs) purified from bone marrow aspirates are being used in the treatment of superficial digital flexor tendon (SDFT) injuries in the horse with promising results. In this study the fate of autologous and allogeneic MPCs following injection into the SDFT was monitored by stable transfection of MPCs with green fluorescent protein (GFP). Small lesions were created manually in one forelimb SDFT of 2 horses and injected with autologous MPCs, allogeneic MPCs or bone marrow supernatant alone. Post mortem examinations performed after 10 or 34 days revealed GFP labelled cells located mainly within injected lesions, but with a small proportion integrated into the crimp pattern of adjacent healthy areas of tendon. Furthermore, there was no visible cell mediated immune response to allogeneic MPCs in either of the host horses.     

Equine embryonic stem‐like cells and mesenchymal stromal cells have different survival rates and migration patterns following their injection into damaged superficial digital flexor tendon

Reasons for performing study: Injury to the superficial digital flexor tendon (SDFT) is common in racing and sport horses and poor tendon regeneration leads to high reinjury rates. Autologous mesenchymal stromal cells (MSCs) are being used clinically to improve tendon regeneration but they have some practical limitations. Embryonic stem cells (ESCs) may overcome these limitations but their fate following injection into the damaged SDFT is unknown. Objective: To inject MSCs and ESCs into distinct areas of damage in the SDFT and monitor their survival over a 3 month period. Methods: MSCs and ESCs expressing different reporter genes were injected into separate sites of mechanically induced damage in SDFTs. Cell survival and distribution were examined post mortem after 10, 30, 60 and 90 days and host immune responses determined. Results: Neither MSCs nor ESCs produced signs of cell‐mediated immune response or tumour formation. ESC survival was high and numbers were maintained at a constant level over 90 days. ESCs were present at all sites of damage. In contrast, MSCs showed <5% survival at 10 days and numbers declined over the course of the experiment. MSCs were detected only at the site into which they were injected. Conclusions: ESCs survived in greater numbers than MSCs in the damaged tendon and did not induce an immune response, or form tumours at the injection sites in the 90 day time period studied. ESCs also demonstrated an ability to migrate to other areas of damage within the same tendon, whereas MSCs did not. Potential relevance: ESCs can be used allogeneically, therefore providing a possible ‘off the shelf’ source of cells for therapeutic use which overcomes the practical limitations of autologous MSCs. Furthermore, MSCs and ESCs have different survival rates and migration patterns in the damaged tendon, suggesting that they may produce different functional effects. This may have clinical relevance to treating tendon injuries in the horse.     

Thromboelastographic Clot Characteristics of Autologous Equine Blood Products After Activation by Autologous Thrombin,Bovine Thrombin,or Calcium Chloride

Objective

To compare clotting efficiency of platelet‐rich plasma (PRP) and concentrated platelet‐poor plasma (cPPP) to citrated whole blood after activation by autologous thrombin, bovine thrombin, or calcium chloride (CaCl₂).

Study Design

Experimental study.

Animals

Healthy adult horses (n = 6).

Methods

PRP and cPPP were prepared by commercial devices. Using thromboelastography, clotting variables were compared after activation of citrated autologous blood, PRP, and cPPP by autologous thrombin, bovine thrombin, or CaCl₂, respectively.

Results

PRP had the greatest clot strength and quickest clot rate, whereas cPPP had the weakest clot strength, slowest clot rate, and longest clot initiation time. Bovine thrombin resulted in the shortest clot initiation time, quickest clot rate, and was similar to CaCl₂ for greatest clot strength. CaCl₂ also resulted in the longest clot initiation time and time to reach maximum clot strength. Autologous thrombin resulted in the lowest clot strength.

Conclusion

When combined with either bovine thrombin or CaCl₂, PRP provided the best combinations for clinical use. Autologous thrombin was suboptimal, but could be an autologous alternative for clinical application. As prepared here, cPPP had inefficient clotting, but may be sufficient for plasma spray indications.     

Subconjunctival bone marrow‐derived mesenchymal stem cell therapy as a novel treatment alternative for equine immune‐mediated keratitis: A case series

Equine immune‐mediated keratitis (IMMK) leads to increased corneal opacity and inflammation secondary to an alteration of the local immune system. Bone marrow‐derived mesenchymal stem cells (BM‐MSC) have been shown to modulate the immune system by downregulating inflammation. Four horses with unilateral IMMK poorly responsive to traditional medical treatments underwent novel, autologous subconjunctival BM‐MSC therapy. Bone marrow was harvested and processed as previously described for equine orthopedic disease. Horses received autologous subconjunctival BM‐MSC injections approximately every 3‐4 weeks for 1‐5 treatments total. Horses were maintained on their current medical treatment regimen throughout the BM‐MSC treatment period. Three horses had a positive response to therapy as demonstrated by an increase in corneal clarity, a decrease in neovascularization and a reduction in surface irregularity. One horse was nonresponsive to therapy. These experimental results demonstrate the safety and potential efficacy of an innovative solution for IMMK.     

The Effects of Interlocking a Universal Hip Cementless Stem on Implant Subsidence and Mechanical Properties of Cadaveric Canine Femora

Objective

To determine if an interlocking bolt would limit subsidence of the biological fixation universal hip (BFX^®) femoral stem under cyclic loading and enhance construct stiffness, yield, and failure properties.

Study Design

Ex vivo biomechanical study.

Animals

Cadaveric canine femora (10 pairs).

Methods

Paired femora implanted with a traditional stem or an interlocking stem (constructs) were cyclically loaded at walk, trot, and gallop loads while implant and bone motions were captured using kinematic markers and high‐speed video. Constructs were then loaded to failure to evaluate failure mechanical properties.

Results

Implant subsidence was greater (P = .037) for the traditional implant (4.19 mm) than the interlocking implant (0.78 mm) only after gallop cyclic loading, and cumulatively after walk, trot, and gallop cyclic loads (5.20 mm vs. 1.28 mm, P = .038). Yield and failure loads were greater (P = .029 and .002, respectively) for the interlocking stem construct (1155 N and 2337 N) than the traditional stem construct (816 N and 1405 N). Version angle change after cyclic loading was greater (P = .020) for the traditional implant (3.89 degrees) than for the interlocking implant (0.16 degrees), whereas stem varus displacement at failure was greater (P = .008) for the interlocking implant (1.5 degrees) than the traditional implant (0.17 degrees).

Conclusion

Addition of a stabilizing bolt enhanced construct stability and limited subsidence of a BFX^® femoral stem. Use of the interlocking implant may decrease postoperative subsidence. However, in vivo effects of the interlocking bolt on osseointegration, bone remodeling, and stress shielding are unknown.     

The role of mesenchymal stem cells in veterinary therapeutics - a review

Adult mammalian tissue contains a population of cells known as mesenchymal stem cells (MSC), that possess the capability to secrete regenerative cytokines and to differentiate into specialised cell types. When transplanted to a site of injury MSC embed in damaged tissue and repair and regenerate the tissue by secreting cytokines. The immuno-privileged and immuno-regulatory capabilities of MSC enhance their therapeutic potential not only in autologous but also allogeneic recipients. Studies have demonstrated the beneficial effects of MSC in the treatment of a variety of clinical conditions including osteoarthritis, tendon injuries, and atopic dermatitis in domestic animals. Studies using animal models have shown promising results following MSC or MSC secretion therapy for induced injury in musculoskeletal and nervous systems and some organ diseases. This review describes the stem cell types relevant to regenerative medicine and the procedures used for isolation, identification, expansion, enrichment and differentiation of these cells. We also review the use of MSC in animal models of disease as well as diseases in the clinical veterinary setting.     

Neurotoxicity of intraneural injection of bupivacaine liposome injectable suspension versus bupivacaine hydrochloride in a porcine model

Objective

To test whether neurotoxic effects of a bupivacaine liposome injectable suspension differ from those of a standard formulation of bupivacaine hydrochloride (HCl) after intraneural injection into the sciatic nerves in pigs.

SCINTIGRAPHIC TRACKING OF MESENCHYMAL STEM CELLS AFTER PORTAL,SYSTEMIC INTRAVENOUS AND SPLENIC ADMINISTRATION IN HEALTHY BEAGLE DOGS

Mesenchymal stem cells have been proposed to treat liver disease in the dog. The objective of this study was to compare portal, systemic intravenous and splenic injections for administration of mesenchymal stem cells to target the liver in healthy beagle dogs. Four healthy beagle dogs were included in the study. Each dog received mesenchymal stem cells via all three delivery methods in randomized order, 1 week apart. Ten million fat‐derived allogeneic mesenchymal stem cells labeled with Technetium‐99m (99mTc)‐hexamethyl‐propylene amine oxime(HMPAO) were used for each injection. Right lateral, left lateral, ventral, and dorsal scintigraphic images were obtained with a gamma camera equipped with a low‐energy all‐purpose collimator immediately after injection and 1, 6, and 24 h later. Mesenchymal stem cells distribution was assessed subjectively using all four views. Pulmonary, hepatic, and splenic uptake was quantified from the right lateral view, at each time point. Portal injection resulted in diffuse homogeneous high uptake through the liver, whereas the systemic intravenous injection led to mesenchymal stem cell trapping in the lungs. After splenic injection, mild splenic retention and high homogeneous diffuse hepatic uptake were observed. Systemic injection of mesenchymal stem cells may not be a desirable technique for liver therapy due to pulmonary trapping. Splenic injection represents a good alternative to portal injection. Scintigraphic tracking with 99mTc‐HMPAO is a valuable technique for assessing mesenchymal stem cells distribution and quantification shortly after administration. Data obtained at 24 h should be interpreted cautiously due to suboptimal labeling persistence.     

Collection of peripheral blood CD34+ progenitor cells from healthy dogs and dogs diagnosed with lymphoproliferative diseases using a Baxter-Fenwal CS-3000 Plus blood cell separator

Background

Canine peripheral blood mononuclear cell (PBMC) apheresis using a Baxter‐Fenwal CS‐3000 Plus automated blood cell separator has not been reported.

Objective

To determine the feasibility and safety of using a CS‐3000 Plus blood cell separator with a small volume separation container holder (SVSCH) and small volume collection chamber (SVCC) to harvest canine PBMCs from dogs weighing <50 kg.

Animals

Eight healthy mongrel dogs and 11 client‐owned dogs in clinical remission for lymphoproliferative diseases (LPD).

Methods

In this prospective study, aphereses were performed using a Baxter‐Fenwal CS‐3000 Plus blood cell separator, with or without recombinant human granulocyte colony‐stimulating factor (rhG‐CSF) treatment.

Results

Aphereses from 6 healthy dogs given rhG‐CSF yielded an average of 1.1 × 10⁷ ± 8.2 × 10⁶ CD34+ cells/kg. Aphereses from LPD dogs given rhG‐CSF yielded an average of 5.4 × 10⁶ ± 3.25 × 10⁶ CD34+ cells/kg (P = .17). Higher hematocrit in both groups of dogs receiving rhG‐CSF correlated with an increased number of CD34+ cells/kg harvested (healthy, P = .04; LPD, P = .05). Apheresis was well tolerated by all dogs.

Conclusions and Clinical Importance

Canine PBMC apheresis using the Baxter‐Fenwal CS‐3000 Plus cell separator with an SVSCH and SVCC is a feasible and safe option for harvesting an adequate number of CD34+ peripheral blood progenitor cells from dogs weighing ≥17 kg for hematopoietic cell transplantation.     

Force Plate Gait Analysis in Doberman Pinschers with and without Cervical Spondylomyelopathy

Background

The most accepted means of evaluating the response of a patient with cervical spondylomyelopathy (CSM) to treatment is subjective and based on the owner and clinician's perception of the gait.

Objective

To establish and compare kinetic parameters based on force plate gait analysis between normal and CSM‐affected Dobermans.

Animals

Nineteen Doberman Pinschers: 10 clinically normal and 9 with CSM.

Methods

Force plate analysis was prospectively performed in all dogs. At least 4 runs of ipsilateral limbs were collected from each dog. Eight force platform parameters were evaluated, including peak vertical force (PVF) and peak vertical impulse (PVI), peak mediolateral force (PMLF) and peak mediolateral impulse, peak braking force and peak braking impulse, and peak propulsive force (PPF) and peak propulsive impulse. In addition, the coefficient of variation (CV) for each limb was calculated for each parameter. Data analysis was performed by a repeated measures approach.

Results

PMLF (P = .0062), PVI (P = .0225), and PPF (P = .0408) were found to be lower in CSM‐affected dogs compared with normal dogs. Analysis by CV as the outcome indicated more variability in PVF in CSM‐affected dogs (P = 0.0045). The largest difference in the CV of PVF was seen in the thoracic limbs of affected dogs when compared with the thoracic limbs of normal dogs (P = 0.0019).

Conclusions and Clinical Importance

The CV of PVF in all 4 limbs, especially the thoracic limbs, distinguished clinically normal Dobermans from those with CSM. Other kinetic parameters less reliably distinguished CSM‐affected from clinically normal Dobermans.     

Isolation and Differentiation of Mesenchymal Stem Cells From Bovine Umbilical Cord Blood

Currently, mesenchymal stem cells (MSCs) are used in veterinary clinical applications. Bone marrow and adipose tissue are the most common sources of stem cells derived from adult animals. However, cord blood which is collected non‐invasively is an alternative source of stem cells other than bone marrow and adipose tissue. Moreover, high availability and lower immunogenicity of umbilical cord blood (UCB) haematopoietic stem cells compared to other sources of stem cell therapy such as bone marrow have made them a considerable source for cell therapy, but MSCs is not highly available in cord blood and their immunogenicity is poorly understood. In this study, the cells with spindle morphology from 7 of 9 bovine UCB samples were isolated and cultured. These mesenchymal stromal cells were successfully differentiated to osteocytes, chondrocytes and adipocytes. In addition, Oct‐4 and SH3 were determined by RT‐PCR assay. It is the first report of isolation, culture, characterization and differentiation of bovine umbilical stem cells.     

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.     

Custom total knee replacement in a dog with femoral condylar bone loss

Objective— To report surgical planning, technique, and outcome of custom total knee replacement (TKR) performed to manage a medial femoral condylar nonunion in a dog.
Study Design— Clinical case report.
Animal— A 3-year-old, 20 kg Karelian Bear Hound.
Methods— Computed tomographic scan of the left pelvic limb was used to build a stereolithography model of the distal portion of the femur. The model was used to create a custom augment to replace the missing medial femoral condyle and a custom stem for intramedullary condylar cemented fixation. The augment and stem were adapted to femoral and tibial components already available. The model was used to rehearse the surgery and then the custom prosthesis was implanted.
Results— Weight bearing returned 8 hours after surgery and improved thereafter. Joint alignment was normal and prosthetic joint motion was 60–165° postoperatively. The dog resumed moose hunting 3 months after surgery. Peak vertical force and impulse of the operated limb measured 17 months after surgery were 65% and 47% of the normal, contralateral limb.
Conclusion— Based on short-term follow-up, cemented canine TKR was successfully achieved for management of a severely abnormal stifle joint.
Clinical Relevance— With further refinement and development of commercially available prostheses, TKR should be possible for canine patients.     

Evaluation of adverse reactions in dogs following intravenous mesenchymal stem cell transplantation

Background

Recent studies have assessed the therapeutic potential and drawbacks of mesenchymal stem cells (MSCs). The adverse reactions of intravenous transplantation of bone marrow (BM)-derived MSCs were examined at varying doses and frequencies of administration.Nine healthy beagle dogs were purchased from a commercial laboratory. The dogs were distributed equally (n = 3 per group) and randomly into three groups. All dogs received allogeneic BM-derived MSCs: 2 × 10⁶ once (group A), 2 × 10⁷ once (group B), and 2 × 10⁶ for three consecutive days (group C). Various laboratory examinations, multi-detector computed tomography features and histopathology were evaluated to clarify the clinical and diagnostic features of adverse reactions of MSCs administration, prior to receiving MSCs (pre procedure) and on days 1, 3, and 7 post transplantation.

Results

Only one dog had clinical signs during and after MSCs transplantation. Dogs receiving 2 × 10⁶ MSCs showed increased numbers of lymphocytes but the total white blood cell counts were not elevated (P < 0.01). Multi-detector computed tomography (MDCT) revealed pulmonary parenchymal changes in one dog and histopathologic examination revealed pulmonary parenchymal edema and hemorrhage in four dogs. The presence of pulmonary thromboembolism was not detected in either examination.

Conclusions

We considered the presence of pulmonary edema and hemorrhage as possible adverse reactions after intravenous MSCs transplantation; however these results should be cautiously interpreted.     

Prevalence of proteinuria in a canine oncology population

Objective

To evaluate the point prevalence of proteinuria in dogs presenting to the University of Georgia Oncology Service for the first time.

Materials and Methods

In this prospective study, 60 client‐owned dogs with a confirmed cancer diagnosis were included but those with lower urinary tract neoplasia were excluded. Each dog's signalment, cancer diagnosis, previous cancer treatments, current medications and travel history were recorded. Renal values, electrolytes, packed cell volume, total solids, systolic blood pressure, urinalysis, urine protein:urine creatinine and retinal examinations were recorded. Non‐proteinuric, borderline proteinuria and overt proteinuria were defined as urine protein:urine creatinine <0·2, ≥0·2 but <0·5, and ≥0·5, respectively. Urine culture was performed in dogs with active urine sediments or overt proteinuria.

Results

Twenty‐nine dogs were non‐proteinuric (48·3%), 22 (36·7%) borderline proteinuric and nine (15%) overtly proteinuric. None were azotaemic. Hypertension (systolic blood pressure ≥160 mmHg) was detected in 18 (30%) dogs. Of these, six were non‐proteinuric, nine borderline proteinuric, and three overtly proteinuric. Proteinuria was detected in 51% of dogs presented to our oncology service, the majority of which were classified as borderline.

Clinical Significance

The high proportion of proteinuria in dogs in this study suggests that screening for proteinuria in dogs with cancer may be prudent. Larger studies are required to correlate specific cancer types and the impact of treatment with the development, magnitude and persistence of proteinuria.     

Primary Cilia in Chondrogenic Differentiation of Equine Bone Marrow Mesenchymal Stem Cells: Ultrastructural Study

Locomotor system disorders in equine species, such as tendon lesions, osteoarthritis, or ligament injuries, are some of the most frequent causes of dramatic reduction in horse performance. Traditional therapies are aimed at the inflammatory process and pain, but they do not regenerate normal tendon or ligament matrix and do not reduce re-injured rates. Mesenchymal stem cells have started to use as therapeutic option to repair these injured tissues. Most studies have focused on their isolation, in vitro culture and phenotyping. However, mesenchymal stem cell ultrastructure has been disregarded in the last years. We investigate the ultrastructural characteristics of these cells once differentiated into chondrocytes. Ultrastructural analysis was conducted on suspension cultures of differentiated chondrocytes from bone marrow mesenchymal stem cells by means of transmission electron microscopy. The morphologic characteristics of these cells, their ability to produce the extracellular matrix, and the presence of a single cilium could be indicative of the mesenchymal cells differentiation into chondrocyte phenotype. This study provides essential data to evaluate the degree of suitable phenotypic stability, for these cells can be used with repair purposes.     

Muscle reorganisation through local injection of stem cells in the diaphragm of mdx mice

Background

The diaphragm is the major respiratory muscle affected by Duchenne muscular dystrophy (DMD) and is responsible for causing 80% of deaths. The use of mechanical forces that act on the body or intermittent pressure on the airways improves the quality of life of patients but does not prevent the progression of respiratory failure. Thus, diseases that require tissue repair, such as DMD, represent a group of pathologies that have great potential for cell therapy. The application of stem cells directly into the diaphragm instead of systemic application can reduce cell migration to other affected areas and increase the chances of muscle reorganisation. The mdx mouse is a suitable animal model for this research because its diaphragmatic phenotype is similar to human DMD. Therefore, the aim of this study was to assess the potential cell implantation in the diaphragm muscle after the xenotransplantation of stem cells.

Methods

A total of 9 mice, including 3 control BALB/Cmice, 3 5-month-old mdx mice without stem cell injections and 3 mdx mice injected with stem cells, were used. The animals injected with stem cells underwent laparoscopy so that stem cells from GFP-labelled rabbit olfactory epithelium could be locally injected into the diaphragm muscle. After 8 days, all animals were euthanised, and the diaphragm muscle was dissected and subjected to histological and immunohistochemical analyses.

Results

Both the fresh diaphragm tissue and immunohistochemical analyses showed immunopositive GFP labelling of some of the cells and immunonegativity of myoblast bundles. In the histological analysis, we observed a reduction in the inflammatory infiltrate as well as the presence of a few peripheral nuclei and myoblast bundles.

Conclusion

We were able to implant stem cells into the diaphragm via local injection, which promoted moderate muscle reorganisation. The presence of myoblast bundles cannot be attributed to stem cell incorporation because there was no immunopositive labelling in this structure. It is believed that the formation of the bundles may have been stimulated by cellular signalling mechanisms that have not yet been elucidated.