Sarah Broeckx

Regenerative Skin Wound Healing in Mammals: State-of-the-Art on Growth Factor and Stem Cell Based Treatments

Mammal skin has a crucial function in several life-preserving processes such as hydration, protection against chemicals and pathogens, initialization of vitamin D synthesis, excretion and heat regulation. Severe damage of the skin may therefore be life-threatening. Skin wound repair is a multiphased, yet well-orchestrated process including the interaction of various cell types, growth factors and cytokines aiming at closure of the skin and preferably resulting in tissue repair. Regardless various therapeutic modalities targeting at enhancing wound healing, the development of novel approaches for this pathology remains a clinical challenge. The time-consuming conservative wound management is mainly restricted to wound repair rather than restitution of the tissue integrity (the so-called “restitutio ad integrum”). Therefore, there is a continued search towards more efficacious wound therapies to reduce health care burden, provide patients with long-term relief and ultimately scarless wound healing. Recent in vivo and in vitro studies on the use of skin wound regenerative therapies provide encouraging results, but more protracted studies will have to determine whether the effect of observed effects are clinically significant and whether regeneration rather than repair can be achieved. For all the aforementioned reasons, this article reviews the emerging field of regenerative skin wound healing in mammals with particular emphasis on growth factor- and stem cell-based therapies.

Regenerative Therapies for Equine Degenerative Joint Disease: A Preliminary Study

Degenerative joint disease (DJD) is a major cause of reduced athletic function and retirement in equine performers. For this reason, regenerative therapies for DJD have gained increasing interest. Platelet-rich plasma (PRP) and mesenchymal stem cells (MSCs) were isolated from a 6-year-old donor horse. MSCs were either used in their native state or after chondrogenic induction. In an initial study, 20 horses with naturally occurring DJD in the fetlock joint were divided in 4 groups and injected with the following: 1) PRP; 2) MSCs; 3) MSCs and PRP; or 4) chondrogenic induced MSCs and PRP. The horses were then evaluated by means of a clinical scoring system after 6 weeks (T1), 12 weeks (T2), 6 months (T3) and 12 months (T4) post injection. In a second study, 30 horses with the same medical background were randomly assigned to one of the two combination therapies and evaluated at T1. The protein expression profile of native MSCs was found to be negative for major histocompatibility (MHC) II and p63, low in MHC I and positive for Ki67, collagen type II (Col II) and Vimentin. Chondrogenic induction resulted in increased mRNA expression of aggrecan, Col II and cartilage oligomeric matrix protein (COMP) as well as in increased protein expression of p63 and glycosaminoglycan, but in decreased protein expression of Ki67. The combined use of PRP and MSCs significantly improved the functionality and sustainability of damaged joints from 6 weeks until 12 months after treatment, compared to PRP treatment alone. The highest short-term clinical evolution scores were obtained with chondrogenic induced MSCs and PRP. This study reports successful in vitro chondrogenic induction of equine MSCs. In vivo application of (induced) MSCs together with PRP in horses suffering from DJD in the fetlock joint resulted in a significant clinical improvement until 12 months after treatment.

Allogenic Mesenchymal Stem Cells as a Treatment for Equine Degenerative Joint Disease: A Pilot Study

Cell-based therapies, such as treatments with mesenchymal stem cells (MSCs) and platelet-rich plasma (PRP) are thought to have beneficial effects on the clinical outcome of orthopedic injuries, but very few animal studies with large sample size are published so far. Therefore, the aim of this study was to assess the safety and report the clinical outcome of allogenic, immature or chondrogenic induced MSCs in combination with PRP for the treatment of degenerative joint disease (DJD) in 165 horses. MSCs and PRP were isolated from a 6-year-old donor horse and transplanted either in their native state or after chondrogenic induction in combination with PRP into degenerated stifle (n=30), fetlock (n=58), pastern (n=34) and coffin (n=43) joints. Safety was assessed by means of clinical evaluation and the outcome was defined as failure to return to work (score 0), rehabilitation (score 1), return to work (score 2) and return to previous level (score 3), shortly (6 weeks) after treatment or at 18 weeks for the patients that returned for long-term follow-up (n=91). No adverse effects were noticed, except for three patients who showed a moderate flare reaction within one week after treatment of the fetlock joint without long-term effects (1.8% of 165 horses). Already after 6 weeks, 45% (native MSCs) and 60% (chondrogenic induced MSCs) of the treated patients returned to work (→ score 2+3) and the beneficial effects of the treatment further increased after 18 weeks (78% for native MSCs and 86% for chondrogenic induced MSCs). With the odds ratio of 1.47 for short-term and 1.24 for long-term, higher average scores (but statistically not significant) could be noticed using chondrogenic induced MSCs as compared to native MSCs. For all three lower limb joints a higher percentage of the treated patients returned to work after chondrogenic induced MSC treatment, whereas the opposite trend could be noticed for stifle joints. Nevertheless, more protracted follow-up data should confirm the sustainability of these joints.

Intravenous Application of Allogenic Peripheral Blood-Derived Mesenchymal Stem Cells: A Safety Assessment in 291 Equine Recipients

It has been reported that mesenchymal stem cells (MSCs) have homing capacities and immunomodulating effects after an intravenous injection. However, transplanting MSCs in murine tail veins can result in pulmonary reactions and even death of the animals. Unfortunately, only a few intravenous MSC transplantations have been reported in large animal species and these were performed in a limited number of individuals. To assess the safety of MSC transplantations, a large study on 291 recipient horses is reported here. MSCs were isolated from the peripheral blood (PB) of a 4-year-old and 6-year-old donor horse after having tested their PB for a wide range of transmittable diseases. The MSC samples from both donor horses were characterized and resuspended in 1 ml of Dulbeccos Modified Eagle Medium (DMEM) supplemented with 10% Dimethyl Sulfoxide (DMSO). After hand-thawing in the field, 291 horses with ages ranging from 3-months to 33-years were directly injected into their jugular vein. 281 horses (97%) received a single injection of a physiological dose of 0.2 x10(6) MSCs, 5 horses (1.7%) were re-injected after approximately 6 weeks (using the same dose and donor cells) and a single superphysiological dose of 10(6) MSCs was administered to 5 horses as well. In total, 176 recipients were injected with MSCs from the 4-year-old donor and 115 recipients received MSCs from the 6-year-old donor. From all the injected horses (n=291) no acute clinical adverse effects were noticed. Apart from one horse that died of colic 7 months after the treatment, no deaths were registered and all the horses were monitored for 1 year after the injection. In conclusion, no adverse effects were noticed in 291 recipients after an intravenous injection of allogenic PBderived MSCs. Nevertheless, further research is warranted in order to verify the immunogenic properties of these cells after allogenic transplantation into various (patho)physiological sites.

The effects of equine peripheral blood stem cells on cutaneous wound healing: a clinical evaluation in four horses.

Stem‐cell therapy represents a promising strategy for the treatment of challenging pathologies, such as large, infected wounds that are unresponsive to conventional therapies. The present study describes the clinical application of peripheral blood stem cells (PBSCs) for the treatment of four adult Warmblood horses with naturally occurring wounds, which were unresponsive to conventional therapies for at least 3 months. A visual assessment was performed, and a number of wound‐healing parameters (granulation tissue, crust formation and scar formation) were evaluated. In all cases, tissue overgrowth was visible within 4 weeks after PBSC injection, followed by the formation of crusts and small scars in the centre of the wound, with hair regeneration at the edges. In conclusion, this is the first report of PBSC therapy of skin wounds in horses, and it produced a positive visual and clinical outcome.

Equine epidermis: a source of epithelial-like stem/progenitor cells with in vitro and in vivo regenerative capacities.

Besides the presence of somatic stem cells in hair follicles and dermis, the epidermis also contains a subpopulation of stem cells, reflecting its high regenerative capacity. However, only limited information concerning epidermis-derived epithelial-like stem/progenitor cells (EpSCs) is available to date. Nonetheless, this stem cell type could prove itself useful in skin reconstitution after injury. After harvesting from equine epidermis, the purified cells were characterized as EpSCs by means of positive expression for CD29, CD44, CD49f, CD90, Casein Kinase 2β, p63, and Ki67, low expression for cytokeratin (CK)14 and negative expression for CD105, CK18, Wide CK, and Pan CK. Furthermore, their self-renewal capacity was assessed in adhesion as well as in suspension. Moreover, the isolated cells were differentiated toward keratinocytes and adipocytes. To assess the regenerative capacities of EpSCs, six full-thickness skin wounds were made: three were treated with EpSCs and platelet-rich-plasma (EpSC/PRP-treated), while the remaining three were administered carrier fluid alone (PRP-treated). The dermis of EpSC/PRP-treated wounds was significantly thinner and exhibited more restricted granulation tissue than did the PRP-treated wounds. The EpSC/PRP-treated wounds also exhibited increases in EpSCs, vascularization, elastin content, and follicle-like structures. In addition, combining EpSCs with a PRP treatment enhanced tissue repair after clinical application.

Tenogenesis of Equine Peripheral Blood-Derived Mesenchymal Stem Cells: In vitro Versus In vivo

Background: Tendon injuries are a major cause of orthopaedic injuries, and often compromise the return to the same performance level. Therefore, different regenerative therapies, such as Mesenchymal Stem Cells (MSCs) and Platelet-rich Plasma (PRP) have been explored to improve tendon healing in horses. However, ectopic bone formation of undifferentiated cells is a major concern, because of reports of this phenomenon, after intralesional injections of MSCs in rabbit Achilles tendons. Methods: After MSC and PRP isolation of the Peripheral Blood (PB) of a donor horse, tenogenic induction of the MSCs (Tendo-Cell®) was confirmed through morphological changes and immunohistochemistry stainings. Secondly, the safety and clinical effects (ultrasound imaging) of Tendo-Cell®, in combination with PRP, was evaluated after a single intralesional injection in the lateral edge of the Superficial Digital flexor tendon (SDFT) (n=10) and the lateral branch of the suspensory ligament (SL) (n=15) in 25 horses. Different independent veterinary practitioners were asked to give a score from 0-5, at approximately 6 weeks after treatment (0=no ultrasonic improvement and 5=no ultrasonic abnormalities, the tendon has the same consistency and fiber orientation as the contralateral tendon). Results: In 96% of the patients, the same positive evolution was noticed on the ultrasound images, and this was translated to a score 3 or more (= 60% improvement or better than a successful conservative therapy). In addition, for both tendons (SDFT & SL), 40% of the horses received a score 5. Conclusion: In conclusion, the present study is the first to describe the treatment of lesions in the SDFT and SL, with allogenic tenogenic induced PB-derived MSCs, in combination with PRP, with a positive outcome in 24 out of the 25 horses.

The Influence of Allogenic Mesenchymal Stem Cells on the Hematological Status of Horses

Background: It has been reported that mesenchymal stem cells (MSCs) have homing capacities after an intravenous injection and immuno modulating effects in vitro. However, there are no reports on their effect on the animal’s hematological status. Methods: Therefore, MSCs were isolated and characterized from a 4-years-old horse donor after having tested his blood on a wide range of transmittable diseases. Then, 6 horses (13- to 15-years-old) received a single injection into the jugular vein: 3 with MSCs and 3 with the carrier fluid consisting of DMEM and 10% of DMSO. Multiple blood samples were taken at different time points at the same time of the day: before the intravenous MSC (Veno-Cell®) injection ( T0) and 1 day (T1), 1 week (T2), 3 weeks (T3) and 2 months (T4) after the treatment. Results: In the control group no considerable changes were noticed over time, whereas, in all the treated horses, the baseline adjusted cortisol levels (P=0.0490) and number of neutrophils (P=0.0042) were significantly higher and glucose levels significantly lower (P=0.033). At time point T1, baseline adjusted blood thrombocyte levels were significantly higher in the treated group as compared to the control group (P<0.0001). On the other hand, baseline adjusted basophils, eosinophils, lymphocytes and monocytes remained approximately the same in all the horses regardless the treatment. Additionally, the levels of total protein, albumin, alkaline phosphatase and all the tested minerals were not influenced by the injections. Conclusion: In all patients consistent alterations in hematological parameters were noticed after intravenous allogenic MSC treatment. However, further research is warranted in order to verify what the exact influence of these changes might be on the animal’s metabolism, general condition and performance ability.

Desmitis of the Accessory Ligament of the Equine Deep Digital Flexor Tendon: A Regenerative Approach

Background: Desmitis of the accessory ligament of the deep digital flexor tendon (ALDDFT) is a very common problem in different breeds of sport horses, with an inability to heal adequately after conservative therapy in many cases. However, the treatment of different kinds of tendon lesions with platelet-rich plasma (PRP), mesenchymal stem cells (MSCs), or even tenogenic induced MSCs have been reported with promising results. Methods: After MSC and PRP isolation of the peripheral blood (PB) of a donor horse, tenogenic induction of the MSCs (Tendo-Cell®) was confirmed, and samples were microbiologically tested and frozen. Adverse and clinical effects (macroscopic noticeable swelling, lameness and ultrasound imaging) of PRP (n=4), or Tendo-Cell® (n=4) were evaluated after a single intralesional injection in the ALDDFT in 8 horses. For each case, two independent veterinary practitioners were asked to give a score from 0-5, at approximately 6 weeks and 4 months after treatment (0=no ultrasonographic improvement and 5=90-100% improvement, or (nearly) no ultrasonographic abnormalities, the ligament has (almost) no hypo-echoic foci, and (nearly) the same fiber orientation as the contralateral ligament). Results: At approximately 6 weeks post injection, the ultrasound images of all 4 horses treated with PRP only slightly improved. Therefore, a score 2/5 was given by the two attending veterinarians to all 4 patients. In the Tendo- Cell® treated group, on the other hand, all 4 horses received a score 4/5 by the same veterinarians at 6 weeks after intralesional injection. This implicates that the lesions improved about 80% on the ultrasound images, which was much better than the 40% improvement in the PRP treated group. Conclusion: This study reports a positive clinical and ultrasonographical outcome after tenogenic induced MSC (Tendo-Cell®) treatment of desmitis of the equine ALDDFT, whereas no considerable improvements could be noticed after treatment with allogenic PRP.

Guidelines to Optimize Survival and Migration Capacities of Equine Mesenchymal Stem Cells

Background: Although mesenchymal stem cell (MSC) therapy is commonly being used in veterinary medicine, no specific guidelines are described to guarantee an optimal cell survival and migration. However, this is indispensable in order to assure an optimal clinical use. Methods: In the present study, equine peripheral blood (PB)-derived MSCs were isolated and characterized. In order to determine the optimal parameters for long-term cryopreservation, the effects of different dimethyl sulfoxide (DMSO) concentrations (5-20%) and thawing methods (temperature-controlled or hand-thawed) on MSC viability were evaluated by means of trypan blue staining. Afterwards, MSC storage was evaluated for 12 months using only 10% of DMSO as a cryoprotectant without rate-controlled freezing. Then, the influence of frequently used anaesthetics and injectable gels on MSC viability was assessed. Finally, the migration capacity of MSCs through hyaluronic acid at different concentrations was assessed. Results: Firstly, we demonstrated that the DMSO concentration and the thawing method had no considerable influence on MSC viability within the 4 hour evaluation after thawing. Secondly, anaesthetics were highly cytotoxic and only 10% of the MSCs survived 3 hours of their presence. Hyaluronic acid- and glycosaminoglycan-based gels were compatible with MSCs and allowed a high cell survival (more than 90%) within 48 hours of follow-up. However, in a polyacrylamide gel, approximately 80% of the MSCs died within 48 hours. Finally, the concentration of the gel influenced the migration capacity of the MSCs. Indeed, only 60% of the MSCs in 20mg/ml of hyaluronic acid attached to the underlying surface within 48 hours, whereas almost 100% of the MSCs in 10mg/ml of the same substance were able to migrate through the gel. Conclusion: The present study reports practical guidelines for the clinical application of equine PB-derived MSCs.