Management of canine wounds using platelet-rich fibrin (PRF) biomaterial. A case series report.

BACKGROUND: The increasing interest in platelet-based therapies has underwritten the development of novel veterinary regenerative treatments. The haemoderivative platelet-rich fibrin (PRF) comprises abundant concentrations of platelets and leucocytes, above the physiologic baseline, which are considered essential elements for wound regeneration, stimulating local angiogenesis, cellular migration, proliferation and differentiation, considered essential for skin repair. OBJECTIVES: This study aimed to describe the treatment of eight dogs with naturally occurring cutaneous wounds, where autologous PRF therapy was applied, using a protocol developed by our group. METHODS: Eight dogs, aged between 7-month and 9-year old, from different breeds and sexes, were enrolled in this study. Four of these wounds were clinically infected. In three cases, two PRF treatments were performed during the first week of treatment, followed by single weekly treatments from the second week onwards, until exophytic granulation tissues were present. In each case, the treatment was finalized only when complete wound closure was achieved. Wounds did not receive topical antiseptics, antibiotics or topical drugs to promote wound healing during the treatment. RESULTS: PRF-grafting treatments were well tolerated in all treated wounds, inducing significant granulation tissue formation. PRF clots acted as a natural tissue filler, promoting epithelization and wound closure, without the requirement of topical antimicrobial/antiseptics application, or additional surgical debridement. Evident skin contraction was recorded in larger injuries and all the treatments resulted in vestigial aesthetic scars where hair growth was also observed. CONCLUSIONS: PRF-therapy obtained promising results, as an alternative wound treatment, revealing a biological regenerative action, prompting the natural skin healing process.

Mesenchymal Stem Cell Studies in the Goat Model for Biomedical Research-A Review of the Scientific Literature.

Mesenchymal stem cells (MSCs) are multipotent cells, defined by their ability to self-renew, while maintaining the capacity to differentiate into different cellular lineages, presumably from their own germinal layer. MSCs therapy is based on its anti-inflammatory, immunomodulatory, and regenerative potential. Firstly, they can differentiate into the target cell type, allowing them to regenerate the damaged area. Secondly, they have a great immunomodulatory capacity through paracrine effects (by secreting several cytokines and growth factors to adjacent cells) and by cell-to-cell contact, leading to vascularization, cellular proliferation in wounded tissues, and reducing inflammation. Currently, MSCs are being widely investigated for numerous tissue engineering and regenerative medicine applications. Appropriate animal models are crucial for the development and evaluation of regenerative medicine-based treatments and eventual treatments for debilitating diseases with the hope of application in upcoming human clinical trials. Here, we summarize the latest research focused on studying the biological and therapeutic potential of MSCs in the goat model, namely in the fields of orthopedics, dermatology, ophthalmology, dentistry, pneumology, cardiology, and urology fields.

Canine-Origin Platelet-Rich Fibrin as an Effective Biomaterial for Wound Healing in Domestic Cats: A Preliminary Study.

Platelet-rich fibrin (PRF) is a recent platelet-based biomaterial, poised as an innovative regenerative strategy for the treatment of wounds from different etiologies. PRF is defined as a biodegradable scaffold containing elevated amounts of platelets and leukocytes having the capability to release high concentrations of bioactive structural proteins and acting as a temporal release healing hemoderivative. This study aimed to evaluate the performance of canine-origin PRF, obtained from blood of screened donors, as a regenerative biomaterial suitable for the treatment of critical wounds in felines. Four short-hair felines with naturally occurring wounds were enrolled in this study. Three of the wounds were considered infected. Each PRF treatment was the result of the grafting of newly produced PRFs at the recipient area. The PRF treatment was initially performed two to three times per week, followed by single weekly treatments. The study was finalized when complete wound closure was achieved. No topical antimicrobial/antiseptic treatment was applied. The present research demonstrated that xenogenic PRFs significantly induced healthy vascularized granulation tissue in lesions with soft tissue deficit, also prompting the epithelization at the injured site. No rejection, necrosis, or infection signs were recorded. Additionally, PRF-therapy was revealed to be a biological cost-effective treatment, accelerating the wound healing process.

Standardized Platelet-Rich Fibrin (PRF) from canine and feline origin: An analysis on its secretome pattern and architectural structure.

Platelet-rich fibrin (PRF) has been incorporated in surgical procedures to promote tissue and bone healing, particularly in human medicine. The rationale for the use of platelet-based products stems from the fact that platelets, after being activated, release growth factors (GFs) and other active molecules such as cytokines, that modulate inflammation and tissue repair. Although PRF has been advanced as a therapeutic treatment for veterinary use, namely in canine and feline patients (following human medicine developments), to our knowledge a full characterization of PRF therapeutic effectors has never been performed. Herein, we studied the biological properties and release profile of GFs and other cytokines throughout ten days in in vitro culture conditions, in order to investigate the potential therapeutic ability of PRF for canine and feline practice. A protocol for obtaining PRF from whole blood without anti-coagulant from both species was optimized, originating large and homogenous PRF clots. Then, PRF clots obtained from four dogs and four cats were incubated in culture medium to assess the temporal release of platelet-derived growth factor-BB (PDGF-BB), vascular endothelial factor-A (VEGF-A), transforming growth factor ß-1 (TGF-ß1), and interleukin-8 (IL-8). Furthermore, morphological characterization of PRF clots, fresh and after 10 days of incubation, was performed by histology and high-resolution field emission electron scanning microscopy. In standard culture conditions, PRF clots from both species released PDGF-BB, TGF- ß1 and VEGF-A, in a sustained manner, up to day 10. Moreover, PRF presents an initial burst release of IL-8, a mediator of inflammatory response which plays a key role in neutrophil recruitment and degranulation. Overall, our findings show that PRF clots may be an efficient therapeutic strategy in canine and feline clinical practice, accelerating the local healing mechanism, through the sustained delivery of signalling molecules involved in the healing cascade.

Clinical application of mesenchymal stem cells therapy in musculoskeletal injuries in dogs-a review of the scientific literature.

Mesenchymal stem cells (MSCs) are multipotent, which is defined by their ability to self-renew while maintaining the capacity to differentiate into a certain number of cells, presumably from their own germinal layer. MSCs therapy is based on their anti-inflammatory, immunomodulatory (immunosuppressive), and regenerative potential. This review aims to provide a clinical overview of the MSCs potential as a therapeutic option for orthopedic diseases in dogs. A total of 25 clinical studies published in the scientific literature in the last 15 years on various diseases will be presented: semitendinosus myopathy, supraspinatus tendinopathy, cruciate ligament rupture, bone fractures and defects, and also osteoarthritis (OA). All articles involved in this study include only diseases that have naturally occurred in canine patients. MSCs therapy in the veterinary orthopedic field has great potential, especially for OA. All studies presented promising results. However, MSCs bone healing capacity did not reveal such favorable outcomes in the long term. Besides, most of these clinical studies did not include immunohistochemistry, immunofluorescence, and histopathology to confirm that MSCs have differentiated and incorporated into the injured tissues. This review summarizes the current knowledge of canine MSCs biology, immunology, and clinical application in canine orthopedic diseases. Despite the positive results in its use, there is still a lack of defined protocols, heterogeneous samples, and concomitant medications used with MSCs therapy compromising therapeutic effects. Further studies are needed in the hope of overcoming its limitation in upcoming trials.

Platelet-Derived Products in Veterinary Medicine: A New Trend or an Effective Therapy?

Platelet-derived products (PDPs) have gained popularity, mainly due to their high concentrations of bioactive molecules such as growth factors and cytokines, which play important roles in tissue healing and regeneration. PDPs are obtained through minimally invasive procedures and their therapeutic effect has been widely recognized. In veterinary medicine, however, the lack of standard protocols to generate PDPs is a major hurdle for assessing the clinical relevance of PDP-based therapies and for their widespread usage. The aim of this review is to analyze the technical and scientific specificities of PDPs in terms of preparation methodologies, classification categorization, nomenclature, and biological proprieties to advance their future biotechnological potential in veterinary contexts.

Evaluation of hematology, general serum biochemistry, bone turnover markers and bone marrow cytology in a glucocorticoid treated ovariectomized sheep model for osteoporosis research.

Osteoporosis is a metabolic disorder characterized by a loss of bone mass and structure and increasing the risk of fragility fractures, mostly among postmenopausal women. Sheep is a recognized large animal model for osteoporosis research. An experimental group of ewes (3-4 years old) was subjected to ovariectomy (OVX) and weekly glucocorticoid (GC) application for 24 weeks and compared with a sham control group. Blood and bone marrow parameters were analyzed before and 24 weeks after OVX and GC administration. Osteopenia was confirmed through micro-computed tomography and histomorphometric analysis of L4 vertebra in the study end. A statistically significant increase was observed in mean corpuscular volume, mean cell hemoglobin and monocytes and a decrease in red blood count and eosinophils (p<0.05). Alkaline phosphatase (ALP), gamma-glutamyl transpeptidase, magnesium and α1-globulin increased, and creatinine, albumin, sodium and estradiol decreased (p<0.05). A slight decrease of bone formation markers (bone ALP and osteocalcin) and an increase of bone resorption markers (C-terminal telopeptides of collagen type 1 and tartrate-resistant acid phosphatase) were observed, but without statistical significance. This study aims to contribute to better knowledge of sheep as a model for osteoporosis research and the consequences that a performed induction protocol may impose on organic metabolism.

Bioengineered surgical repair of a chronic oronasal fistula in a cat using autologous platelet-rich fibrin and bone marrow with a tailored 3D printed implant.

Clinical summary: A tissue engineering approach was used to aid the surgical repair of a chronic oronasal fistula (ONF) in a 13-year-old cat. A three-dimensional (3D) printed mesh, tailored to the size and shape of the ONF, was created to support a soft tissue flap used to close the defect; and also to provide a matrix for mesenchymal stromal cells present in bone marrow aspirate and bioactive cytokines and growth factors present in platelet-rich fibrin harvested from the patient. A CT scan at day 75 after surgery revealed the formation of new tissue in the defect and the healing process was complete at follow-up 6 months after surgery. Relevance and novel information: Complications are frequently reported following surgical repair of ONFs and include dehiscence of the palatal suture line, flap necrosis due to damage to the greater palatine artery and maxillary osteomyelitis, mainly due to chronic infection and bone lysis. The case described here demonstrates how input from a multidisciplinary team and the use of a biomaterial, processed by sophisticated technologies, can create a precision regenerative medicine strategy adapted to the patient's clinical needs; this provided a novel therapeutic solution for an otherwise hard to treat clinical problem.

Large Animal Models for Osteochondral Regeneration.

Namely, in the last two decades, large animal models - small ruminants (sheep and goats), pigs, dogs and horses - have been used to study the physiopathology and to develop new therapeutic procedures to treat human clinical osteoarthritis. For that purpose, cartilage and/or osteochondral defects are generally performed in the stifle joint of selected large animal models at the condylar and trochlear femoral areas where spontaneous regeneration should be excluded. Experimental animal care and protection legislation and guideline documents of the US Food and Drug Administration, the American Society for Testing and Materials and the International Cartilage Repair Society should be followed, and also the specificities of the animal species used for these studies must be taken into account, such as the cartilage thickness of the selected defect localization, the defined cartilage critical size defect and the joint anatomy in view of the post-operative techniques to be performed to evaluate the chondral/osteochondral repair. In particular, in the articular cartilage regeneration and repair studies with animal models, the subchondral bone plate should always be taken into consideration. Pilot studies for chondral and osteochondral bone tissue engineering could apply short observational periods for evaluation of the cartilage regeneration up to 12 weeks post-operatively, but generally a 6- to 12-month follow-up period is used for these types of studies.

Assessment of bone healing ability of calcium phosphate cements loaded with platelet lysate in rat calvarial defects.

Injectable calcium phosphate cements have been used as a valid alternative to autologous bone grafts for bone augmentation with the additional advantage of enabling minimally invasive implantation procedures and for perfectly fitting the tissue defect. Nevertheless, they have low biodegradability and lack adequate biochemical signaling to promote bone healing and remodeling. In previous in vitro studies, we observed that the incorporation of platelet lysate directly into the cement paste or loaded in hyaluronic acid microspheres allowed to modulate the cement degradation and the in vitro expression of osteogenic markers in seeded human adipose derived stem cells. The present study aimed at investigating the possible effect of this system in new bone formation when implanted in calvarial bilateral defects in rats. Different formulations were assessed, namely plain calcium phosphate cements, calcium phosphate cements loaded with human platelet lysate, hybrid injectable formulations composed of the calcium phosphate cement incorporating hyaluronin acid non-loaded microparticles (20% hyaluronin acid) or with particles loaded with platelet lysate. The degradability and new bone regrowth were evaluated in terms of mineral volume in the defect, measured by micro-computed tomography and histomorphometric analysis upon 4, 8 and 12 weeks of implantation. We observed that the incorporation of hyaluronin acid microspheres induced an overly rapid cement degradation, impairing the osteoconductive properties of the cement composites. Moreover, the incorporation of platelet lysate induced higher bone healing than the materials without platelet lysate, up to four weeks after surgery. Nevertheless, this effect was not found to be significant when compared to the one observed in the sham-treated group.

Exploring the Potential of Starch/Polycaprolactone Aligned Magnetic Responsive Scaffolds for Tendon Regeneration.

The application of magnetic nanoparticles (MNPs) in tissue engineering (TE) approaches opens several new research possibilities in this field, enabling a new generation of multifunctional constructs for tissue regeneration. This study describes the development of sophisticated magnetic polymer scaffolds with aligned structural features aimed at applications in tendon tissue engineering (TTE). Tissue engineering magnetic scaffolds are prepared by incorporating iron oxide MNPs into a 3D structure of aligned SPCL (starch and polycaprolactone) fibers fabricated by rapid prototyping (RP) technology. The 3D architecture, composition, and magnetic properties are characterized. Furthermore, the effect of an externally applied magnetic field is investigated on the tenogenic differentiation of adipose stem cells (ASCs) cultured onto the developed magnetic scaffolds, demonstrating that ASCs undergo tenogenic differentiation synthesizing a Tenascin C and Collagen type I rich matrix under magneto-stimulation conditions. Finally, the developed magnetic scaffolds were implanted in an ectopic rat model, evidencing good biocompatibility and integration within the surrounding tissues. Together, these results suggest that the effect of the magnetic aligned scaffolds structure combined with magnetic stimulation has a significant potential to impact the field of tendon tissue engineering toward the development of more efficient regeneration therapies.

Evaluation of the composition of Carica papaya L. seed oil extracted with supercritical CO2.

Among the most important tropical fruit grown in the world today and in Brazil, papaya occupies a prominent place. Native to tropical America, papaya has spread to several regions of the world, and Brazil accounts for 12.74% of the world production, followed by Mexico, Nigeria and India. The culture reached a harvested area of 441,042 ha and production of 12,420,585 t worldwide. The largest interest in this fruit relies on its main constituent compounds, like vitamins A, B and C, alkaloids (carpaine and pseudocarpaine), proteolytic enzymes (papain and quimiopapain) and benzyl isothiocyanate, more known as BITC, which has anthelmintic activity. Because of that, the present work has as objective the evaluation of the efficiency and composition of the oil extracted from Carica papaya L. seeds with supercritical carbon dioxide. The experiments were performed in a unit containing mainly a high-pressure pump and a stainless steel extractor with 42 mL of volume. The sampling was performed at each 20 min until the saturation of the process. About 6.5 g of sample were fed for each experiment done at 40, 60 and 80 °C under the pressures of 100, 150 and 200 bar. Samples of the Carica papaya L. fruit were acquired in a popular market and free for personal use intended for the study. After collection, the seeds were crushed with the help of a pestle, and dried at 60 °C for 60 min. For each operational condition, the extraction curves were constructed relating cumulative mass of oil extracted in function of the operational time. The better efficiencies were found at 40 °C and 200 bar (1.33%) followed by 80 °C and 200 bar (2.56%). Gas chromatography and NMR analysis could identify an insecticide component (BITC) that enables new applications of this residue in pharmaceutical and chemical industries.

Electrospinning of Bioinspired Polymer Scaffolds.

Electrospinning is a technique used in the production of polymer nanofibre meshes. The use of biodegradable and biocompatible polymers to produce nanofibres that closely mimic the extracellular matrix (ECM) of different tissues has opened a wide range of possibilities for the application of electrospinning in Tissue Engineering. It is believed that nano-features (such as voids and surface cues) present in nanofibre mesh scaffolds, combined with the chemical composition of the fibres, can stimulate cell attachment, growth and differentiation. Despite the widespread use of electrospun nanofibres in tissue engineering, the present chapter will focus on the advances made in the utilisation of these materials in bone, cartilage and tooth related applications. Several aspects will be taken into consideration, namely the choice of polymers, the surface modification of the nanofibres in order to achieve mineralisation, and also the biological application of such materials.

Evaluation of the in vitro and in vivo biocompatibility of carrageenan-based hydrogels.

Carrageenans are highly sulphated galactans, well-known for their thermogelation properties which have been extensively exploited in food and cosmetics industry but poorly explored in the biomedicine field. In this study, we have assessed the in vitro and in vivo biocompatibility of κ-carrageenan hydrogels that have been explored for regenerative medicine and tissue engineering applications. The in vitro cytotoxicity of the materials using a L929 mouse fibroblast cell line was evaluated, and the effect of κ-carrageenan hydrogels on the activation of human polymorphonuclear neutrophils cells (hPMNs) was also evaluated by the quantification of reactive oxygen species by chemiluminescence. Subsequently, the inflammatory/immune reaction to κ-carrageenan hydrogels on subcutaneous implantation was studied in rats. Explants were retrieved after 1 and 2 weeks of implantation for histological and RT-PCR analysis. The cytotoxicity screening revealed that κ-carrageenan hydrogels did not significantly affect L929 metabolic activity. Moreover, hPMNs contact with κ-carrageenan resulted in a reduced and a neglectable signal regarding the detection of superoxide and hydroxyl anions, respectively. The results from the in vivo experiments indicated that κ-carrageenan induce a low inflammatory response. Overall, the data obtained suggest that κ-carrageenan hydrogels are biocompatible and thus can be further studied for their use in target biomedical applications.

Undifferentiated human adipose-derived stromal/stem cells loaded onto wet-spun starch-polycaprolactone scaffolds enhance bone regeneration: nude mice calvarial defect in vivo study.

The repair of large bony defects remains challenging in the clinical setting. Human adipose-derived stromal/stem cells (hASCs) have been reported to differentiate along different cell lineages, including the osteogenic. The objective of the present study was to assess the bone regeneration potential of undifferentiated hASCs loaded in starch-polycaprolactone (SPCL) scaffolds, in a critical-sized nude mice calvarial defect. Human ASCs were isolated from lipoaspirate of five female donors, cryopreserved, and pooled together. Critical-sized (4 mm) calvarial defects were created in the parietal bone of adult male nude mice. Defects were either left empty, treated with an SPCL scaffold alone, or SPCL scaffold with human ASCs. Histological analysis and Micro-CT imaging of the retrieved implants were performed. Improved new bone deposition and osseointegration was observed in SPCL loaded with hASC engrafted calvarial defects as compared to control groups that showed little healing. Nondifferentiated human ASCs enhance ossification of nonhealing nude mice calvarial defects, and wet-spun SPCL confirmed its suitability for bone tissue engineering. This study supports the potential translation for ASC use in the treatment of human skeletal defects.

Chondrogenic potential of two hASCs subpopulations loaded onto gellan gum hydrogel evaluated in a nude mice model.

Cells constitute one of the fundamental components of any cartilage tissue engineering approach. Adipose tissue derived stem cells (ASCs) have a promising future considering the abundance of this tissue in the human body, ease of harness, and the high number of stem cells that can be isolated from small amounts of tissue. However the stromal vascular fraction of the adipose tissue that is isolated upon digestion by collagenase followed by a rough selection of the adherent cells, is composed of many different types of cells, some of which may compromise the proliferation and the differentiation of the ASCs. This manuscript reports a study on the in vivo chondrogenic potential of two ASCs specific subpopulations isolated using a method based on immunomagnetic beads coated with specific antibodies. These ASCs subpopulations, isolated using immunomagnetic beads coated with CD29 and CD105 antibodies, were subsequently transfected with green fluorescent protein (GFP), expanded, and pre-differentiated into the chondrogenic lineage, before being encapsulated in a novel hydrogel based on gellan gum, that has recently been showed to promote in vitro and in vivo cartilage tissue formation. The two ASCs subpopulations encapsulated in the gellan gum hydrogel and in vitro pre-differentiated, were then subcutaneously implanted in nude mice for 6 weeks. Explants were analyzed by various techniques, namely histology, immunohistology and real time RT-PCR that demonstrated the different behaviour of the two ASCs subpopulations under study, namely their potential to differentiate into the chondrogenic lineage and to form new cartilage tissue.

Xenofree enzymatic products for the isolation of human adipose-derived stromal/stem cells.

Human adipose-derived stromal/stem cells (ASCs) are an abundant, readily available population of adult stem cells that reside in adipose tissue and that have a great potential utility for tissue engineering and regenerative medicine therapeutic applications. Several preclinical studies have shown that ASCs have therapeutic applicability, but a standardized isolation and expansion methodology for clinical cell therapy has yet to be established. ASC are typically isolated and expanded using reagents with xenogenic components and this may pose certain risks and safety issues, such as exposure to infectious agents and immune reactions, creating further obstacles to the translation of ASC-based therapies to clinical scenarios. The objective of this study was to determine the suitability and efficacy of various alternative enzymatic products, CLS1 (Worthington), CLSAFA (Worthington), NB4 (SERVA), and Liberase (Roche), for the digestion of adipose tissue and subsequent isolation of ASCs, assessing cell functionality concerning their proliferation and differentiation ability. Results show that there are no statistically significant differences on yield and proliferation of cells isolated after enzymatic digestion with any of the studied products. The differentiation potential of the cells was not affected, and cell surface marker expression was similar among all products. We concluded that clinical grade products can replace current research-grade products effectively in our cell isolation protocols without any negative effect in the yield or function of human ASCs.

The effect of storage time on adipose-derived stem cell recovery from human lipoaspirates.

Multipotent adipose-derived stromal/stem cells (ASCs) can be isolated with high yield from human subcutaneous lipoaspirates. This study reports our experience isolating, expanding, differentiating and immunophenotypically characterizing ASCs over a period of 4 days after having surgically obtained the lipoaspirate samples. The ultimate goal is to understand how to optimize the consistent isolation of ASCs from lipoaspirates. The length of time between adipose tissue harvest and processing will need to be systematically evaluated with respect to cell yield, viability, and function since some distance is likely to exist between the plastic surgeon's office where lipoaspiration is performed and the current Good Manufacturing Practices (cGMP) laboratory where the ASCs are isolated. The objective of this study was to determine the effect of time delays on the yield and function of ASCs after collagenase digestion. We were able to isolate ASCs from lipoaspirates up to 72 h after the surgical procedure. The ASCs isolated on sequential days after the original tissue harvest proliferated, differentiated and maintained cell surface markers. We found that the initial 24-hour period is optimal for isolating ASCs with respect to cell yield and that there was no significant difference between ASC cell proliferation and differentiation ability within this period of time. In contrast, each of these parameters declined significantly for tissues maintained at room temperature for 48 or 72 h prior to isolation. These findings should be considered in the future development of standard operating procedures for cGMP processing of clinical-grade human ASCs.

Use of animal protein-free products for passaging adherent human adipose-derived stromal/stem cells.

Adherent adipose-derived stromal/stem cells (ASC) have been used in pre-clinical regenerative medical studies applied to a broad range of tissues with an ultimate goal of translating these findings to clinical safety and efficacy testing; however, many protocols passage the cells using porcine-derived trypsin. We have compared porcine trypsin with animal protein-free products from recombinant bacteria (TrypLE Express; Invitrogene) and corn (TrypZean; Sigma) based on cell yield, viability and immunophenotype. ASC harvested with each trypsin product were comparable.