The use of stem cells in veterinary especially in fracture healing in dogs

Background

Bone fractures are one of the most common clinical conditions encountered in animals in Sri Lanka. There are two ways of fracture healing namely, primary healing and secondary healing. Primary bone healing involves intra-membraneous ossification while secondary bone healing involves endochondral ossification. Fracture healing involves a complex and sequential set of events to restore the bone , in which stem cells play a crucial role.(Aiyer, no date). Bone defects present a therapeutic challenge to both the surgeon as well as to the patient. When not treated properly, patients can have long-lasting symptoms such as non-unions, pseudoarthrosis and large bone defects. Such patients are left untreated due to the absence of further facilities to enhance bone growth and fracture healing. Tissue engineering with stem cell auto grafts have proven to be the gold standard for fracture healing and therefore is used by surgeons to treat bone defects and large-bone fractures. Bone grafts are considered to be the second most transplanted tissues, exceeded only by blood(Nguyen, 2013)

 

Stem cells are undifferentiated and immortal type of cells which could be transformed to any kind of specialized cells(Deb, 2015). The existence of non-haematopoietic stem cells in bone marrow was reported by the German pathologist one hundred and thirty seven years ago. He injected an insoluble aniline dye into the veins of animals and observed the appearance of dye-containing cells in wounds at a distal site. and indicated that most cells originated from the bone marrow(Fayaz et al., 2011).

Even though there is no exact definition of mesenchymal stem cells, the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy has proposed minimal criteria to define human MSCs, which is thought to be applicable to animals as well:

·         MSC must be plastic-adherent & MSC must express CD105, CD73 and CD90 & MSC lack expression of CD45, CD34, CD14, CD11b, CD79a, CD19 and HLA-DR surface molecules

·         MSC must differentiate to osteoblasts, adipocytes and chondroblasts in vitro

·      MSC lack of expression of haematopoietic Antigen

(Fayaz et al., 2011)

 

At present the use of stem cells have gained wide popularity in the biomedical field due to their high potential of differentiation (Vidal, 2008;Pacitti, 2006; Human et al., 2014; Gomes et al., 2017). Stem cells are a promising and growing field in the area of research in the medical field (Akpancar et al., 2016). Although there is much literature in the use of stem cells in preclinical set-ups there is limited literature in the use of stem cells clinically in animals. 

1.                  Stem cells

Stem cells are immortal and undifferentiated cells that can differentiate into specialized cells(Deb, 2015).

Types of stem cells:

1.                  Embryonic stem cells

First described by James Thompson in 1988. These are obtained from the blastocyst during in-vitro fertilization. These can be easily transplanted but the rejection risk is higher.

2.                  Mature stem cells

First described in the 1960’s. Obtained from body tissues, umbilical cord or placenta after birth and are classified as multi-potent cells.

Stem cells could also be categorized into 4 according to differentiating potential namely, totipotent cells which are only present in embryo, pluripotent cells which can differentiate into any cell, multi-potent cells which can differentiate into a limited type of cells and uni-potent cells which are the mature form.(Akpancar et al., 2016)

Regardless of the cell type, the overall process of transforming the cell is alike. The adult stem cell will first be harvested from the original and will have copies of four embryonic genes inserted into the cells to make them like embryonic stem cells. After the insertion of these genes, the cells then become induced pluripotent stem cells. The cells then require a specific environment in order to differentiate into the desired cell type. Each of the different environments required will need a different type and amount of certain growth factors in order to replicate conditions within the body. After the cells have differentiated and are in large enough amounts, they can then be grafted into the body of the patients(Riggs, 2017)(American Veterinarian, 2018)

1.1.1.      Potential of stem cells

Stem cells can differentiate into more than 200 types of specialized cells. Stem cells primarily produce progenitor cells which have more specialized functions such as brain cells and red blood cells(Akpancar et al., 2016).Since stem cells are immortal they can be used to generate cell lines that can be amplified to generate large numbers of cells.(Deb, 2015) Also, due to their capability of differentiation, they can generate heart cells for someone possessing a damaged heart, brain cells for a Parkinson’s or Alzheimer’s patient, liver cells for a hepatitis patient, or nerve cells for spinal cord injuries(Deb, 2015). Stem cells are a promising field of research in the medical field(Akpancar et al., 2016)

The gold standard for defining the potential of stem cells for differentiation is by in vivo transplantation under defined experimental conditions(Bianco P, Riminucci M, Gronthos S, 2001).

Embryonic stem cells have the ability to differentiate into any cell in the body. Some have argued that adult stem cells  obtained from bone marrow or umbilical cord blood should be pursued instead of embryonic stem cells because they believe the derivation of stem cells from embryos is ethically unacceptable(Johnson, 2007).

Mesenchymal stem cells are found in periosteum, bone marrow, muscle, fat and synovium which can differentiate into osteoblasts, chondrocytes, tenocytes,  adipocytes and myoblasts(Fossum, no date). Adipose tissue provides a relatively abundant supply of MScs and is easy to be isolated in large numbers than compared to bone marrow.(Shah et al., 2018) adipose tissue is ideal for same day harvest and injection, making it attractive for traumatic injury cases(Qureshi et al., 2018)

Properties of MSCS:

·         Anti-inflammatory and immunosuppressive abilities that help to significantly attenuate the immune responses in the host. These cells along with soluble bioactive com- pounds are then able to inhibit T lymphocyte, B lymphocyte and natural killer cell activation(Shah et al., 2018).

According to Felix and Rosa on a study of mesenchymal stem cell-organized bone marrow elements: an alternative haematopoietic resource BMMSCs are capable of forming ectopic bone and associated BM structures when transplanted into immune-compromised mice subcutaneously using hydroxyapatite tri-calcium phosphate (HA/TCP) as a carrier vehicle. Bone formation would start at 4 weeks post-transplantation along with a significant in-growth of blood vessels in the interstitial compartments. By 6-8 weeks post-transplantation BM structure emerges around established bone (Felix and Rosa, no date). In a similar study there is evidence that even in a normal fracture healing tibia model; MSC transplant promotes the repair process supporting the use of MSC to provide a critical number of regenerative cells that are necessary for fracture healing. 

Factors affecting the growth of MSCs:

o   biomaterial scaffold

o   cell biology techniques

o   growth factors

·         vascular endothelial growth factor-repair heart failure

·         IGF – wound healing

·         Epidermal growth factor – wound healing

·         Hepatocyte growth factor-amelioration of multiple sclerosis

o   mechanical environment

o   exosomes

(Hospital, 2012) (Miller et al., 2006) (Mankani et al., 2006)

In a study conducted on translational clinical establishing Criteria for human mesenchymal stem cell potency the in vitro characterization of MSCs was followed by a functional in vivo assay to determine the ability of high- and low- growth capacity MSCs to contribute to the formation of ectopic bone in mice over an 8-week period. It was found that MSCs from all donors has a capacity to form ectopic bone despite the various extents of scaffold seeded with high growth capacity. MSCs showed a greater ability to form ectopic bone than that with both scaffold alone or scaffold seeded with low-growth capacity MSCs and there is no difference between scaffold alone and scaffold-seeded with low-growth MSC. To date, no systematic correlation between the properties of MSCs in vitro and their in vivo efficacy is known because there is no universally accepted in vitro method for predicting the therapeutic potency of MSCs.(Group et al., 2015)

1.1.2.      Current applications of stem cells

The use of stem cell therapy in animals came into use among veterinarians first in 2004(American Veterinarian, 2018).Studies on the benefits of stem cells have so far been mostly positive in the research models.(Riggs, 2017)

Key determinants in selection of an appropriate MSC source include the accessibility of the harvest tissue, stem cell population density, and ease of cell differentiation(Qureshi et al., 2018)

Adult mesangioblast cells have proven promising results in the treatment of muscular dystrophy(Deb, 2015).  MSC injections may be an effective adjunct in the management of osteoarthritis and a variety of cartilage related pathologies(Qureshi et al., 2018). Stem cells are mainly used to treat chronic inflammatory diseases where excessive bone loss is commonly observed, changes at the bone surface may reflect changes within the bone marrow.(Fierro, Nolta and Adamopoulos, 2017) Research efforts have focused on spinal cord injury, multiple sclerosis, Parkinson’s disease, Alzheimer’s disease, diabetes, and other diseases or conditions.(Shimabukuro, 2007). The first successful bone marrow transplant that resulted in long term survival of a patient suffering from leukemia was performed by Dr. Donnall Thomas in 1956(Bhojwani, 2006) Treatment of Diabetes with Adult Pancreatic Stem Cells(Bhojwani, 2006) Treatment of Parkinson’s disease Using Adult Neural Stem Cells Parkinson’s(Bhojwani, 2006)

Clinical trial based on the use of mesenchymal stem cells from autologous bone marrow in patients with lumbar intervertebral degenerative disc disease(Human et al., 2014). In both veterinary and human medicine, stem cell therapy has been studied in varied conditions, including cancer, heart disease, immune disorders, Alzheimer and Parkinson diseases, blindness, and diabetes. One of its most widespread uses is in bone marrow transplant. In the veterinary world, stem cell therapy has been used primarily in soft tissue injury and wound healing. (American Veterinarian, 2018)

In equine medicine stem cell therapy is mostly used to treat overstrain injuries of the palmar metacarpal tendons and ligaments. In one of the largest studies of stem cell therapy, 113 race-horses with an injury to the superficial digital flexor tendon demonstrated a re-injury rate of 27% after bone marrow–derived mesenchymal stem cell therapy, which was statistically significantly lower than the re-injury rate in historic controls (50%-60%).The findings showed that 2 years after allogeneic tenogenically induced mesenchymal stem cell therapy was administered 5 days after injury, more than 80% of horses returned to their previous level of performance, and re injury rates were 18% compared with 44% for conventional treatments(American Veterinarian, 2018).

 A study of 93 client-owned dogs with osteoarthritis was conducted at sites across the United States. After 2 months, dogs treated with allogeneic adipose tissue–derived mesenchymal stem cells exhibited greater improvements in owner-assessed activity and greater decreases in veterinary-assessed pain compared with dogs that received placebo treatment. In another randomized study of 39 dogs with hip osteoarthritis, intra-articular adipose tissue-derived mesenchymal stem cell therapy improved function and quality of life, with results maintained at 6 months. However, other studies have shown less permanent results.(American Veterinarian, 2018)

Cell therapy has been very successful as a treatment option for various diseases and could revolutionize veterinary medicine in the future. Bone marrow stem cells are promising in the medical field, but there are still some difficulties to be faced in relation to the handling, differentiation and application of these cells in vivo(Gomes et al., 2017).

Future applications of stem cells may include direct implantation and/or ex vivo tissue engineering, in combination with biocompatible/biomimetic biomaterials and/or natural or recombinantly derived biologics. MSCs could also be used in gene therapy applications for the delivery of genes or gene products and tissue banks. Future studies should be directed to find out the  cellular and molecular characteristics for optimal identification, isolation, and expansion, and to understand the natural, endogenous roles of MSCs in normal and abnormal tissue functions(Pacitti, 2006) . There is limited research on ex vivo conditions such as the  composition and structure of the carrier or even the volume of cells required for regeneration of a volume of a bone.(Bianco P, Riminucci M, Gronthos S, 2001)

One of the latest stem cell types are the pathenotes. These are obtained by  electrically stimulating egg cells to divide to the blastocyst stage where the inner mass cells are then taken and an ES cell line is established.(Deb, 2015)

1.1.3.  Obstacles of stem cell therapy

The main obstacles with the use of stem cells are its cost, the need of a well trained staff and the difficulty in access due to the lack of facilities. Stem cell therapy is still in its initial stage, and positive outcomes cannot be guaranteed and may even be temporary for many that undergo the procedure. The American Veterinary Medical Association has issued a position statement that encourages the study of stem cell therapy but cautions its use in routine clinical practice. After stem cell therapy the pet should rest for 10 days and have restricted activity for 30 days  (American Veterinarian, 2018).

Auto grafts are bone tissue retrieved from the patient’s own bone through a
surgical extraction procedure, most commonly at the iliac crest. It is thought that transplantation of the bone will provide viable tissue with biological function; however, the removal will damage the cellular components, tissue continuity, and the tissue’s regenerative ability. Compared to allografts and donor bone tissue, auto grafts increase patient risk during extraction procedures but have enhanced graft-to-tissue integration (osteo integration) and compressive strength. The auto graft harvesting causes complications in 8-20% of all patients, including blood loss, nerve damage, artery damage, chronic pain, tissue necrosis, and infection [4]. For patients with bone disease, auto graft extraction and implantation significantly increases healing complications due to decreased bone quality and regeneration ability; hence, allografts are the alternative of choice.

Allograft is donor bone tissue that has been processed to remove all cellular, bacterial and viral components to eliminate immune response and disease transmission [7]. Even though processing significantly compromises osteo genic and mechanical properties of the tissue, it is the material of choice for 35% of all grafting procedures because of its availability, shelf-life, and customizable type and size [8]. The physiochemical properties of allografts are different for fresh, frozen, or freeze-dried allografts. Fresh allografts are rarely used because of the extended time required for screening to prevent disease transmission(Nguyen, 2013)(Thompson et al., 2015)

MSC Cellular and biochemical factors related to chondro genesis are other key developing areas of research. Long-term safety and efficacy of these therapies has not been proven(Qureshi et al., 2018)

As mentioned previously, the location of the adult stem cell can be a deciding factor in the outcome of the results. The success of this type of method is very dependent upon the type of cell being used. From each location within the body, cells will have different needs for their regeneration. Since adult stem cells are used, the most common way to use them is to create the induced pluripotent stem cells as they can then become many different types of cells rather than the certain niche they occupied. Based on these original locations though, the cell will Have different genes and conditions such as molecules, growth factors, and other markers which will also help in the future differentiation of the cell as adult stem cells seem to have somewhat of a memory of their history (Riggs, 2017)
 important issue for the success of any stem cell therapy is the quality of the cells administered, with regard to cellular integrity, age, senescence, proliferative and differentiative capacity, and intact paracrine function. a number of clinical studies have shown that cell preparations, including bone marrow, bone marrow concentrates, and ex vivo cell preparations, can be efficiently used to facilitate bone healing in non-unions, critical‐sized segmental defects, or osteonecrosis(No, Press and Emtam, 1975)

It was also discussed that the methods are not ideal for everyone as they do have their limitations such as a person’s age and the injury itself. Problems can be furthered by the cells themselves and their “memories” in the induction of these cells into their desired cell types(Riggs, 2017)

There was a sex‐difference in the rate of fracture healing(Readmissions and Program, 2016a)

Although there are controversies, legal issues with regard to the use of stem cells, there is no prohibition in its use despite a few restrictions in some countries.(Shimabukuro, 2007)

The optimal number of MSCs needed for transplantation is also unknown.(Serigano et al., 2010)

the availability of MSCs is limited as a result of the comorbidity and complications associated with MSC harvest from adipose tissue or bone marrow.(Martins-taylor and Xu, 2011)

All patients exhibited significantly improved bone volume with no side effects. bone tissue regeneration represents an important challenge for oral‐maxillofacial surgeons, dentists, and orthopedic and plastic surgeons.(Barton Laws et al., 2013)

2.                  Bone fractures

2.1.1.      Methods of fracture healing

Disorders in the musculoskeletal system are painful and are one of the global health problems. Current research focuses on identifying an effective way of reducing morbidity associated with musculoskeletal injuries(Akpancar et al., 2016). 

Fracture healing is the biologic process after cartilage and bone disruption has occurred and is no longer able to maintain its continuity for proper functioning. The goals of fracture treatment are to promote healing, restore function of affected bones and tissues and to obtain a cosmetically acceptable appearance.(‘Small_Animal_Surgery.pdf’, no date)

After a fracture there are two main ways of fracture healing namely, endochondral ossification and intra-membraneous ossification. Long bones are formed by endochondral ossification while flat bones are formed by intramembranous ossification.(Leslie, 2016)(Aiyer, no date). Indirect bone healing occurs in fractures with an unstable mechanical environment because of the movement of the adjacent bones. Direct bone healing takes place when a fixation device is placed to maintain absolute fragment stability at the fracture site. Sequential radiographs allow the evaluation of fracture healing. Development of periosteal callus indicates indirect bone formation. Direct fracture healing is indicated by the filling of stable fracture lines with bones. Trabecular bone healing in metaphyseal fractures appears in radiographs as the formation of dense bands at the fracture site(‘Small_Animal_Surgery.pdf’, no date).

3.                  Stem cells in fracture healing

Bone grafts are the second most transplanted tissue which is second to blood. Currently over 500,000 bone grafts are performed in the united states annually.(Nguyen, 2013)

The transplanted grafts are considered necrotic tissues that serve as the template for bone regeneration. The bone healing and repair reactions start with the formation of a hematoma to induce revascularization and recruit progenitor bone cells to the site of injury within 2 weeks. Bone cells then form new woven bone to stabilize and establish skeletal continuity at the fracture, which can take 6 weeks to 6 months. The woven bone is eventually remodeled into mature lamellar bone, years following the implantation [5, 6]. The rate and success of bone repair and regeneration depends on the quality and type of grafts transplanted.(Nguyen, 2013)

The first documented bone tissue engineering attempt was in 1668(Nguyen, 2013).Mesenchymal stem cells are the most widely used cell type for bone regeneration(Guo et al., 2015)(Thompson et al., 2015).

Stem cell therapy has recently become popular especially in the field of orthopaedics(Akpancar et al., 2016).In this area, the first stem cell study was performed by Masquelet and then by Henrich(Akpancar et al., 2016).

Despite the more invasive techniques such as surgeries performed at present, stem cells offer a new treatment option which could bring quicker relief and healing to the areas in need because they are made of cells and are able to replicate quickly(Riggs, 2017).Stem cells are site specific(Readmissions and Program, 2016b).Bone marrow stromal cells are progenitors of skeletal tissue components such as bone, cartilage, the hematopoiesis‐supporting stroma, and adipocytes(Bianco P, Riminucci M, Gronthos S, 2001).More than 20% of dogs undergo bone fractures and out of that 5-20% of the total fractures result in non-unions. Despite the prevalence of fractures that result in non-unions and delayed unions there is no effective means of resolving such cases.(Leslie, 2016)

Cell-based therapies using adipose-derived stem cells (ASCs) provide one possible form of treatment for bone defects. ASCs are multi potent, easily isolated, and readily available. (Leslie, 2016).

The technique by which cortical or cancellous bone is transplanted is known as bone grafting.  Bones transplanted from one site to another in the same animal is called auto genous bone grafting. Auto genous bone grafting allows fracture healing before implants fail. Auto genous bone grafts are histocompatible. Allografts are bones transplanted from one animal to the other of the same species. Cellular antigens of these grafts could be recognized as antigens resulting in graft rejection. Allo-implants are bones treated by chemical preservation, irradiation, and autoclaving or freezing to remove the cellular activity. Composite grafts are allografts reinforced with cancellous grafts. Xeno-grafts are bones transplanted from one species to another(‘Small_Animal_Surgery.pdf’, no date). Bone grafts can be sources of osteo progenitor cells that help in osteo genesis. They provide various degrees of mechanical support. The type of bone graft selected is determined by the function required to optimize fracture healing. Cancellous auto grafts are highly cellular but mechanically weak thus providing a good osteo conductive, osteo inductive and osteo genic capability but less substantial fracture support. Cortical allo implants have the exact opposite capabilities to that of cancellous auto implants. Auto genous bone grafts are often used due to its ease of harvest, availability and osteo genic properties and are recommended to assist healing when optimal healing is not expected especially during delayed unions, non-unions and cortical defects after fracture repair.(‘Small_Animal_Surgery.pdf’, no date)

According to Bianco, Riminucci and Grinthos on a study conducted about stem cells the local transplantation of marrow stromal cells for therapeutic applications allows the efficient reconstruction of bone defects larger than those that would heal naturally.

Several pre-clinical studies on animal studies have shown the feasibility of marrow stromal cells grafts for ortho paedic purposes.(Bianco P, Riminucci M, Gronthos S, 2001). Bone marrow osteo genesis at the site of graft depends between individuals and between species. Marrow osteo progenitor cells combined with demineralized  bone matrix was effective in stimulating bone formation In a canine model of a gap non-union as open autologous cancellous bone grafting(‘Small_Animal_Surgery.pdf’, no date).

Complications of bone grafting include seldom pain at the donor site, infection, graft rejection, failure of fracture repair and graft fracture, seroma formation at donor site, wound dehiscence at donor site but complications at recipient site are difficult to be recognized.(‘Small_Animal_Surgery.pdf’, no date).

Cancellous bone auto graft is the gold standard to against which all types of bone grafts are compared(Fossum, no date)(Thompson et al., 2015). Different types of bone grafts are auto grafts, allografts, biomaterials(demineralized bone matrix, collagen),synthetic bone substitutes(tri calcium phosphate ceramics, bio glass and polymers) and composites of osteo genic  cells, osteoinductive growth factors and synthetic osteoconductive matrix.(Fossum, no date)

Sources of stem cells used for orthopaedic procedures include Bone Marrow derived- Mesenchymal Stem Cells (BM-MScs), Adipose Derived Mesenchymal Stem cells (AD-MScs), Peripheral Blood-Derived Progenitor Cells, Synovial Tissue-Derived Stem Cells (ST-MSC) and Bone marrow concentrate. 

              -BM-MScs: bone is the most commonly used stem cell source that can differentiate into muscular skeletal cells. This method is quite painful to the patient

            - AD-MScs: more reliable source of MScs less invasive, considerable amount of proliferation and provides maximal strength to serum-deprivation induced apoptosis.

            - ST-MSC:  used in cartilage repair procedures

            - Bone marrow concentrate: This procedure was proposed by Buda et al. (32) and

Gobbi et al. (33) and the iliac crest are used for this technique. Bone marrow is aspirated and then centrifuged. (Akpancar et al., 2016)

AD-MSCs and BM-MSCs have been most preferred  sources of MSCs; however, there is a controversy about the most effective source of MSCs for orthopedic procedure(Akpancar et al., 2016).

Autologous cell-seeded scaffolds are good candidates for tendon injury repairs.(Theses, 2013)

One of the earliest studies to assess the in vivo potential of cartilage constructs for bone formation used a distinct in vivo model with the excision of an entire bone (Huang et al., 2006). The authors investigated the potential of an autologous cartilage construct derived from MSCs for carpal bone reconstruction following lunate excision in the rabbit forelimb. This was the first time that whole-bone repair was attempted and the study demonstrated that the cartilage construct was capable of supporting woven bone tissue formation following 12 weeks of implantation. Moreover, the study also demonstrated presence of neo-vascularization following implantation demonstrating the potential of such cartilage-based systems to promote blood vessel ingrowth.(Thompson et al., 2015)

Contrastingly, in a study conducted by Shan et al. on the outcome of allogeneic adult stem therapy in dogs suffering from osteoarthritis and other joint defects none of the dogs had displayed any adverse effects other than slight discomfort after undergoing adipose derived MScs; two of the dogs had exhibited mild skin allergy that had been managed by anti-allergic medication(Shah et al., 2018).

According to Khosla percutaneous autologous bone marrow grafting was a potentially effective and safe method for the treatment of atrophic tibial diaphyseal nonunion(Khosla, Westendorf and Mödder, 2010).

4. Assessing the outcome of the therapy

Estimation of bone formation and bone union will be assessed in vitro by semi-quantitatively scoring the extent of bone within each transplant on a logarithmic scale by independent individuals.

In vivo transplants will be scored on a scale of 0-4; a score of 0 corresponds to no bone formation where as a score 4 corresponds to abundant bone formation greater than one half of the section.

The degree of bony union between transplant and adjacent bone will be evaluated grossly by attempting to move the transplant manually and observing the extent to which it could be displaced from its original location. A union will be scored on a scale of 0-4; a score of 0 corresponds to the absence of any union between transplant and adjacent mouse bone, whereas a score of 4 will be given to transplants that are grossly secure and where bony union was evident in greater than one half of the section.

In addition, follow up of the dog after about seven weeks for radiographs of the fractured segment will be performed to assess the outcome of the therapy. 

5.  Expected outcome

After a stem cell transplant to a bone fracture we do expect a more rapid growth of the bone defect compared to the time taken for bone healing after an intra-medullary bone pinning. In addition greater bone stability is to be expected than compared to the bone stability acquired after healing by bone pinning. Better results are also anticipated with the transplant of stem cells into bone disorders such as bone mal-union and bone non-union.

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