First Published: 17 November 2016
AbstractClinical aspects of using Adipose Derived Stem Cells to treat Intervertebral Disc Disease are discussed. A two-pronged therapeutic plan is described which involves treating disc degeneration and damaged spinal cord with stem cells. Stem cell treatment offers prophylactic options for discs that have not yet ruptured and therapy, both alone and combined with standard surgical techniques. Injection of stem cells into the nucleus pulposus of the degenerating disc causes a rapid regeneration of the matrix and rehydration of the disc. This injection is facilitated using C-Arm Radiology. In addition, the effective outcomes of hemilaminectomy surgery are doubled by utilizing Adipose Derived Stem Cells injections into the spinal parenchyma during the surgery procedure.1 Successful outcome is based on access to MRI and C-arm radiography, surgical technique, in-house processing of stem cells, and rehabilitation.
IntroductionIntervertebral disc disease (IVDD) is a serious disease of the vertebral column affecting the spinal cord, causing pain and neurologic damage. IVDD can affect all dogs but is predominantly a disease of dogs with chondrodystrophy. Chondrodystrophic pets are literally those with “bad cartilage growth,” including but not limited to the Dachshund, Corgi, Cocker Spaniel, Shih Tzu, Basset Hound, and Beagle. In the United States, there are approximately 300,000 dachshunds. Over 25% of dachshunds will suffer from IVDD during their lifetime, resulting in 75,000 pets requiring some form of treatment for IVDD.
Intervertebral Disc Disease Anatomy and PathophysiologyThe vertebral column supports the neck and back and is composed of bony vertebral bodies connected by cushioning intervertebral discs. The vertebral canal is the hollow space formed by the vertebral bodies occupied by the spinal cord. The spinal cord is the nerve bundle responsible for all nerve impulses to and from the body.
The normal intervertebral disc (IVD) cushion is shaped like a jelly donut. The interior “jelly” is made of a rich extracellular matrix that forms the central gelatinous nucleus pulposus, which contains cartilage-like cells. This “jelly” is contained by the rest of the “donut” or annulus fibrosus, made of tougher fibroblast-like cells. This cushioning jelly donut structure is connected to the adjacent bone of the vertebral bodies by cartilaginous endplates, Figure 1.
The “jelly” consists of collagen and proteoglycans which are large molecules capable of attracting and retaining water. Functionally, the collagen provides shape and tensile strength, while proteoglycans confer tissue viscoelasticity, stiffness, and resistance to compression, through their interaction with water. A proper balance between matrix synthesis and degradation is responsible for the maintenance of the integrity of the IVD and hence the mechanical behavior of the IVD itself. This is related to the activity of cellular signaling molecules such as cytokines, growth factors, enzymes, and enzyme inhibitors that act to modulate the action of the cells (and stem cells) that make the collagen and proteoglycans. Degeneration of the structure of the annulus fibrosus and the nucleus pulposus occur early in life in chondrodystrophic breeds resulting in shape and molecular changes in the IVD. Progressive degeneration results in loss of water from the “jelly” in the disc and eventual expulsion of the dehydrated “jelly” from the disc resulting in compression of the spinal nerves and spinal cord itself. Intervertebral Disc Disease (IVDD) occurs when the cartilage disc deforms or ruptures causing pressure and or damage to the spinal cord, Figure 2.
Figure 2: Intervertebral Disc Disease: Spinal nerve root and spinal cord compression caused by extruded nucleus pulposus. The disc space is narrower, causing folding and dorsal impingement of the spinal cord by the dorsal longitudinal ligament.
As the intervertebral disc degenerates we see dehydration and tears of the disc annulus and nucleus pulposus. At a molecular level, we see decreased cell viability and proteoglycan synthesis and reduced diffusion of nutrient and waste products. This leads to accumulation of dead cellular debris, degraded matrix macromolecules, and an increased degrading enzymatic activity along with a modification of the collagen distribution.2 It is at this level that stem cells function to regenerate healthy matrix and reform the normal shape of the IVD.
Spinal cord damage from IVDD results from outward to inward compression of the spinal cord. The spinal cord neurons are organized with the less important neurons on the outside and the more important ones on the inside. The outer neurons conduct proprioceptive messages that tell the location and position of the limbs to the brain. The innermost tissues of the spinal cord contain nerve tracts responsible for relaying information from the brain to the muscles. The grey matter of the spinal cord contains nerve cell bodies responsible for spinal reflexes and for coordination. Initial damage to the spinal nerves results in pain as a primary sign, as compression becomes more severe, loss of proprioception is evident, followed by loss of coordination, followed by loss of motor control, then finally loss of deep pain perception. Localization of damage to one side of the spinal cord is evident by comparing the degree of damage and loss of nerve function on one side versus the other. Figure 3.
Figure 3: Spinal Nerve Tracts: Nerve bundles to and from the brain are organized into tracts. These tracts are compressed during IVDD resulting in loss of their function. Depending on the location and severity of the compression, different functions are lost allowing prognostic decisions to be made.
Central nervous system regeneration is highly limited after injury. Spinal cord injury (SCI) leads to cell death, particularly in neurons, oligodendrocytes, astrocytes, and precursor cells. Any cavities and cysts resulting from this cell death and loss may interrupt healing of axonal tracts. SCI culminates in glial scarring, a multifactorial process involving reactive astrocytes, glial progenitors, microglia, macrophages, fibroblasts, and Schwann cells.3 Such scars are often oriented across the neuron path and contain transmembrane molecular inhibitors of axon growth, and appear impenetrable. Lack of regenerative capacities of adult spinal cords results from neuron growth inhibitors of myelin proteins, glial scar formation and a poorly defined extracellular matrix-derived factor.3 Stem cells regenerate the spinal cord by differentiating into astrocytes and oligodendrocytes, as well as neuronal cells. Neurons derived from engrafted stem cells may relay signals from disrupted fibers in the host, including local circuit interneurons or ascending fibers that are present in the dorsal column and grey matter. This neuronal transdifferentiation (changing from one cell type to another based on chemical and molecular by-products) process that occurs with adipose derived stem cells results from the interactions of cells, cytokines provided by these cells, growth factors, and intercellular signals. Adipose derived stem cells have been shown to secrete multiple angiogenic (blood vessel growth) and anti-apoptotic (anti cell death) cytokines that support tissue regeneration and minimize tissue damage. Engrafted stem cells and the chemical factors released by the spinal cord injury play important roles in the proliferation, migration and differentiation of existing spinal cord stem cells. The adipose derived stem cells that survive produce large amounts of fibroblast growth factor and vascular endothelial growth factor in the host spinal cord. These cytokines have been shown to promote neurogenesis.
Adipose Tissue Derived Mesenchymal Stem Cell TherapyTissue engineering and regenerative medicine techniques offer long-term tissue repairing options with the possibility to restore intervertebral disc function. Stem cells have the potential to repopulate and regenerate the IVD. There are several potential sources of stem cells, including adipose tissue, bone marrow, and other tissues such as embryonic and fetal stem cells. Embryonic and fetal stem cells are pluripotent cells with a potential to differentiate into any body tissue. These cells are highly flexible since they can differentiate into any type of tissue, but both the procurement of embryonic stem cells and research are limited, given the related ethical issues. Embryonic cells are not used clinically in the treatment of pets. Figure 4.
These cells can be obtained from multiple adult tissues, including bone marrow, trabecular bone, articular cartilage, muscle, and adipose tissue, which represent a variant of the adult stem cell, Figure 5.
While a variety of cell sources in the field of stem cell research have been proposed for clinical application, Autologous Adipose Stem Cells (ASCs) as a cell source in bone and cartilage repair have gained significant attention. In fact, adipose tissue is considered a suitable source of stem cells for clinical use, given the ease of the procedure to retrieve adipose tissue. This procedure is minimally invasive and large numbers of cells are obtained. Treatment of IVDD can occur with an injection of autologous adipose stem cells (ASCs). The adipose derived cells can be mixed with Platelet Rich Plasma to add growth factors and increase potency. Hyaluronic acid can also be used to provide a support matrix for the stem cells.5
Adipose derived stem cells injected into collapsed, degenerated discs act to regenerate the disc. The stem cells produce a collagen, proteoglycan, and macromolecule matrix that is indistinguishable from a normal disc.This chemical structure attracts water, causing the collapsed disc to re-inflate and expand the disc space between the vertebrae. This flattens out the previously folded ligaments on the floor of the spinal canal, thereby reducing pressure on the spinal cord. The extruded disc material pressing against the spinal cord now has more space and less pressure on it. The anti-inflammatory effects of the ASCs and stromal vascular fraction of cells reduces damaging effects of the previously extruded disc material on the spinal cord. Disc regeneration is rapid, occurring within two weeks of treatment and continuing for at least six months after therapy. Disc regeneration can also be given to patients before complete degeneration and collapse of the disc space to prevent disease associated with compression of the spinal cord.
ASCs injected into damaged spinal cord, aid in healing by reducing the glial scar formation and promoting neurogenesis. Studies have shown that in dogs with no deep pain perception prior to surgical decompression with hemilaminectomy that received a single injection of ASCs into the spinal cord had better clinical outcomes than dogs with surgery alone.1 Improved clinical signs after transplantation of ASCs are primarily attributed to a modified inflammatory environment and increased survival of endogenous nerve cells. Moreover, transplanted ASCs reduce regulatory signal molecules related to glial scar formation and can partially differentiate into neural cells.
Procedures and Methods
Clinical cases of IVDD present at different stages of the disease and so require customizing of therapy. Some pets present with mild behavioral abnormalities such as reluctance to jump, climb or descend stairs, or painful or stiff back or neck. Some pets have mild to moderate proprioception deficits. Other pets have ataxia, crossing of the limbs and reluctance or inability to stand while the most severe pets have no pain perception and are tetraparetic or quadriplegic. Almost all pets are candidates for stem cell therapy as a prevention or treatment with or without surgical decompression. However, the best decisions are made after complete neurologic examination and imaging. MRI imaging is the preferred modality because not only can compression of the spine be perceived but also the amount of water within the disc is an indication of disc health and can be seen on T2 weighted MRI images.5 –Figure 6 & Figure 9. CT images have proven adequate when MRI is not available, and CT scans outperform myelogram studies when evaluating IVDD.6Autologous Adipose Derived Stem Cells (ADCs) are obtained from the patient by harvesting the fat filled falciform ligament through a 2cm midline abdominal incision anterior to the umbilicus. The fat is transferred to a sterile container and processed in a biological safety hood, Figure 7. The fat is minced, washed with PBS and mixed with collagenase type I then incubated, agitated, and centrifuged at 37°C to separate fat from the Stromal Vascular Fraction (SVF). The SVF contains mesenchymal stem cells, preadipocytes, endothelial progenitor cell, T cells, B cells, and mast cells, as well as adipose tissue macrophages. The SVF is filtered through a 100 μm pore filter then filtered again through a 60 μm pore filter. The cells are counted and a ratio of live cells to dead cells is used to calculate live cell doses. In a separate process, blood from the patient is harvested for the creation of Platelet Rich Plasma (PRP).7 The stem cells are mixed with PRP to dilute them and stimulate them to release cytokines. The PRP is activated with autologous thrombin and mixed with the SVF in the proper proportions for administration of 100 μl volume containing 1 x 107 live cells/ml. Individual luer-lok syringes are prepared containing 100 μL stem cell mixture for each disc to be injected. This process takes about 2 hours. The pet is prepared for general anesthesia and the area of skin over the site is surgically prepared. A C-Arm radiology unit is used to properly guide 24ga spinal needles into the disc space. Figure 10. Positioning is confirmed at opposing 90° angles. The stem cells are injected into the center of the nucleus pulposus and the needle is then removed. Figure 11. For the animals who have significant neurologic disease, hemilaminectomy surgery is recommended with stem cell therapy as an adjunct to the surgery and to treat other discs that may be affected. The surgical technique for hemilaminectomy is the standard removal of the bony wall lateral to the spinal cord. Disc material is removed with small atraumatic instruments, Figure 13. The spinal cord is then injected with stem cells. A syringe is prepared with pure platelet rich plasma enriched adipose derived stem cells containing 150 μL volume with 1 x 107 stem cells.1 These cells are injected using a 24GA spinal needle through the dura matter in three locations of 50μL each at the location of damage and above and below the area of damage.
In animals with spinal cord damage as indicated on physical examination or diagnostic imaging, but that are not receiving decompressive surgery, the stem cell and PRP mixture is injected sub-arachnoid much the same as if doing myelography using the cisterna magna or lower lumbar access areas. Total volume of stem cell mixture should be 150 μL per kg and contain 1 x 107 cells per ml.
Physical therapy is an important part of the post-operative recovery plan. The day after the procedure, the pet should be receiving spinal proprioceptive inputs in the form of spatial alterations using foam or inflatable “peanuts” to roll the pet back and forth. Limb placement and massage also stimulate ascending neurons. Figure 14. When neurons damaged by IVDD are stimulated they will release cytokines when the nerve impulses reach the damaged area. These cytokines influence the genetic neurogenesis of the stem cells enabling more accurate repair. Utilizing electrostimulation, massage, or passive range of motion exercises for paraparetic or quadraparetic patients enables spinal tracts to receive information destined for the brain.8 Mental stimulation using the pet’s name, treats and other encouragements creates impulses along the cerebrospinal tracts also liberating cellular chemical signals that communicate to the newly engrafted stem cells. The importance of these chemical messengers in the instruction of stem cell development cannot be overemphasized. Two to three times a day the pet’s spinal and centrally mediated reflexes need to be stimulated using different and alternating modalities. Strength training with water treadmill needs to be started as soon as possible, taking into consideration the need to protect a surgical wound from the water.
DiscussionRegenerative therapy modalities using Adipose Derived Stem Cells are clinically relevant. Stem cells from fatty tissue that does not require culture and expansion is revolutionary. Previously stem cells were derived from bone marrow which was then cultured to increase stem cell numbers over three to six weeks. The next generation of stem cell treatments involved sending fat samples to outside labs which processed the cells and sent them back with no way of the clinician knowing how many live cells were in the final cell sample. Stem cells that can be processed within a couple of hours and are autologous with little if any chance for adverse reactions represent a paradigm shift for all of medicine. Stem cell therapy for neurologic damage to the spinal cord resulting in loss of deep pain perception has been shown to double the success rate.9 This doubling of pets that have complete return to function over surgery alone is a substantial reason that every pet with neurologic disease should have stem cells as an adjunct to surgical therapy. The intervertebral disc nucleus pulposus matrix of molecules is largely devoid of cells with cells counts less than 6000 cells per mm2. Very few cells are required to produce the matrix.4 In fact, too many cells added to the nucleus pulposus matrix will cause death of the cells. The matrix attracts and holds water molecules resulting in a change in the structure of the disc. The disc expands, and this expansion reduces pressure on the spinal cord and restores normal function to the disc. This regeneration of the disc is also paradigm shifting, as many of our previous therapies were designed to destroy the disc by fenestration, laser ablation, etc. Today we can rebuild it within days.
This novel approach must be holistic, encompassing all aspects of diagnosis, treatment, post-operative care and rehabilitation.
The following are considered essential skills or assets in this regard:
- MRI is an essential tool to determine the health of the intervertebral disc. When surgery is anticipated, CT scans or Myelograms are great but they cannot tell you the amount of water in a disc. Disc water content is a key indicator for disc degeneration.
- Surgery capability is still an essential tool for those pets with severe compressive disease.
- Stem Cell Processing Laboratory with biological safety cabinet, cell counting, cell viability, cryopreservation and culture capacity. You must know how many live cells you are placing into the tissues. For example, too many cells in the nucleus pulposus will not live.
- C-Arm Fluoroscopy for accurate placement of stem cells into the nucleus pulposus. This can be done without a C-Arm using anatomical guidelines, skill, touch and many many radiographs to confirm needle location but it is not feasible or advisable for most patients.
- Rehabilitation is essential starting the day after the procedure if possible.
AuthorDr. Garner is the owner and Chief of Staff at Safari Veterinary Care Centers where he has practiced for over 30 years. He continuously thrives to educate clients, pet owners and veterinarians alike about the regenerative, holistic opportunities offered by stem cell therapy. Dr. Garner can easily be reached by your preferred method of contact; by phone at the hospital: 281.332.5612, by email: firstname.lastname@example.org or by filling out our Email Inquiry Form (below).
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