STEM-CELL TREATMENT IN DISC DEGENERATION : WHAT IS THE EVIDENCE ?

Revisar o potencial papel das celulas-tronco para o tratamento de doenca degenerativa do disco intervertebral (IVD). Foi realizada uma revisao dos trabalhos da base de dados Medline em relacao a celulas-tronco e doenca degenerativa discal (DDD). Para fins de discussao dos dados, os trabalhos foram divididos em: revisao, in vitro, experimentais e clinicos. Os tratamentos disponiveis atualmente focam basicamente na reducao dos sintomas, e nao na reversao do processo degenerativo do DIV. O uso de celulas-tronco mesenquimais (CTM) vem sendo proposto como uma opcao de tratamento para DDD. Estudos in vitro demonstraram que as CTM sao capazes de se diferenciarem em celulas do NP e que as CTM tambem diminuem os niveis inflamatorios do DIV degenerado. Alem disso, estudos experimentais demonstraram que as CTM permaneciam viaveis quando injetadas no DIV e que eram capazes de regenerar parcialmente do DIV degenerado e sua estrutura. Os poucos estudos clinicos presentes na literatura apresentam resultados divergentes. O uso de CTM esta sendo amplamente estudado e mostra resultados promissores para o tratamento da DDD. Embora muitos avancos venham sendo alcancados em estudos in vitro e experimentais, ainda faltam estudos clinicos para comprovar o papel das CTM no manejo da DDD.


ResUMen
Revisar el papel potencial de las células madre en el tratamiento de la enfermedad degenerativa del disco intervertebral (IVD).Se realizó una revisión de los artículos de la base de datos Medline sobre células madre y la enfermedad degenerativa del disco (DDD).Para fines de discusión de los datos, los trabajos se dividieron en: revisión, in vitro, experimentales y clínicos.Los tratamientos disponibles actualmente enfocan básicamente la reducción de los síntomas, y no la reversión del proceso degenerativo del IVD.El uso de células madre mesenquimales (MSC) se propone como una opción de tratamiento para DDD.Estudios in vitro demostraron que las MSC son capaces de diferenciarse en células NP y que las MSC también reducen los niveles inflamatorios de la IVD degenerado.Además, estudios experimentales demostraron que las MSC se mantuvieron viables cuando inyectadas en el IVD y que eran capaces de regenerarse parcialmente del IVD degenerado y su estructura.Los pocos estudios clínicos presentes en la literatura presentan resultados divergentes.El uso de MSC se está estudiando ampliamente y muestra resultados prometedores en el tratamiento de la DDD.Aunque se hayan logrado muchos avances en estudios in vitro y experimentales, aún faltan estudios clínicos para comprobar el papel de las MSC en el manejo de  inTRODUcTiOn Degenerative disc disease (DDD) is a common and natural process in the human spine.This degeneration occurs progressively and can affect the biomechanics, stability, and neurological function of the spine 1 .In the United States (US) almost 300.000surgeries are performed every year due to DDD, with the costs related to the surgical procedures rising to 50 billion dollars annually 2 .Overall, 1.5 million disc surgeries are performed worldwide every year 3 .Annually, approximately 5 of 1000 persons will present sciatica due to disc disorders 4 , which are the major cause of chronic low-back pain in the modern world 5 .
Nowadays, degeneration of the intervertebral disc (IVD) is regarded as an irreversible phenomenon.The treatment options involve basically non-surgical (physical therapy, nerve root blocks) and surgical strategies (disc excision and arthrodesis), which are effective only in symptomatic relief and may actually accelerate the degenerative process in adjacent levels 6 .Recent studies demonstrated the potential of stem cell treatment to regenerate or repopulate the degenerated IVD [7][8][9] .
The goal of this study is to review the potential of stem cells as a treatment option for DDD, discussing the actual role of the stem cell treatment.

Physiopathology of Disc Degeneration
The human spine is composed by 23 IVD that separate the vertebrae and provide flexibility.They account for 20 to 30% of the length of the spine and increase in size on progression from the cervical to the lumbar spine 10 .In addition to flexibility the IVD have the function of stability and support loads during exercise.The IVD structure consists basically in a central part called the nucleus pulposus derived from notochord, which is surrounded by the annulus fibrosus derived from mesenchymal tissue 10 .The nucleus pulposus (NP) is rich in proteoglycan and water whereas the annulus fibrosus is rich in collagen, especially type I, II, VI and IX 11,12 .
The composition of the IVD varies with the level of the spine, the collagen content in the nucleus being highest in the cervical discs and lowest in the lumbar discs, whereas the proteoglycan content shows the opposite trend 13 .
In the mature NP, the proteoglycan aggregates are important to retain water and restricting the water flux influences the response of the tissue to loads delivered in the spine 11 .With ageing principally in the nucleus there is a decrease of proteoglycan aggregates and an increase of nonaggregating aggrecan because of proteolytic degradation 14,15 .Thus, with age the ability of resist compressive loading declines.
In the annulus fibrosus the collagen fibrils are oriented in sheets around the NP.The key function of this structure is to retain the NP, taking up and distributing the load exerted by this tissue during various types of exercise 11 .
During life many changes occur in the composition of the extracellular matrix including notochordal cell loss, mesenchymal cell senescence, loss of the vasculature and calcification of the vertebral endplates, which alter or decrease the synthetic capabilities of the disc cells 10 .Nuclear cell density decreases with age throughout life, whereas annular cellularity reaches a plateau after the age of 50 years 16 .The normal turnover capability is impaired in the disc matrix and degeneration products accumulate 10 .
The IVD degeneration and its structural changes result from this continuum catabolism occurring in the extracellular matrix associated with the incapacity of replace the impaired collagen fibers and aggrecan with new molecules during life.The IVD appears incapable of intrinsic repair in the adult, but it is the dream of physicians to be able to induce such biologic repair 17 .

Cell-Based Therapy for Intervertebral Disc Degeneration
Nowadays, surgical and non-surgical therapies to manage IVD degeneration focus only on symptom relief.In recent years, as the understanding of the molecular and cellular events involved in disc degeneration has improved, the idea of manipulating the cellular content of the disc to achieve a beneficial outcome has grown 6,18 .
One treatment option is the use substances in order to stimulate the existing disc cells to increase production of proteoglycan.However, due to the relative accelularity of the degenerated disc it may not be sufficient to achieve a good result.An option for that issue is to introduce in the degenerated disc cells capable of producing appropriate matrix in an attempt to recover the biomechanical properties of the disc 6 .
Nishimura et al. 19 performed the transplantation of autologous NP tissue into denucleated rat discs, where it was shown to slow the progression of degeneration.However, this cell source has practical limitations in the clinical setting due to the necessity of damaging an adjacent disc, likely inducing degeneration in this level.By contrast, adult mesenchymal stem cells (MSC) are easily procured by bone marrow aspiration, and are more capable of adapting successfully to the environment of the disc and therefore of achieving a differentiated state appropriate for long-term matrix synthesis 6 .

Characteristics of Mesenchymal Stem Cells
MSC are undifferentiated, multipotent cells that have the ability to differentiate into a number of cell types, including bone, cartilage, fat, muscle and tendons, depending on the environment and biologic signals provided.It has already been established that MSC can be pushed into chondrogenic or perhaps even discogenic pathways and are capable of expressing aggrecan and Type II collagen in large quantities 20 .Another advantage is the fact that MSC can be obtained from many autologous sources including bone marrow and fat without significant morbidity or immunogenic response and can be easily expanded in cultures 21 .

In Vitro Studies with Stem Cells and Disc Degeneration
It was already known that MSC were able to differentiate into chondrocyte-like cells 22 .Due to the fact that NP and chondrocytes have similar characteristics, it is reasonable to assume that MSC could also be able to differentiate into NP-like cells 23,24 .
Risbud et al. 12 demonstrated that rat MSC when exposed to hypoxia and transforming growth factor-β are able to differentiate into a phenotype consistent with that of the NP via mitogen-activated protein kinase signaling pathways.Using different methods, other authors also demonstrated that MSC are able to differentiate into NP-like cells that could be used in cell-based tissue engineering therapies for IVD regeneration [25][26][27][28][29][30][31][32] .
Blanco et al. 33 performed the isolation and characterization of MSC from human degenerated disc.They found that the degenerated NP contains MSC and that those cells are extremely similar to those found in the bone marrow.These findings suggest that DDD could be treated by cell therapy, injecting differentiated cultured MSC into NP-like cells, and stimulating the MSC already present in the NP.
One of the possibilities to explain why some patients with DDD present more pain than others is the development of intradiscal inflammation.Bertolo et al. 34 studied the immunosuppressive influence of the MSC in fragments of the IVD of patients with DDD.They found that 70% of the patients had a reduction in IgG production and the proliferation of peripheral blood lymphocytes was also reduced when MSC were present.This effect in reducing inflammation shows that the potential role of MSC for DDD goes beyond the ability to repopulate the IVD.

Experimental Studies with Stem Cells and Disc Degeneration
After demonstrating that MSC are able to differentiate into NP-like disc cells, in vivo studies were necessary to evaluate the efficacy and safety of this therapeutic option in degenerated discs.
Crevensten et al. 35 studied the viability of MSC injected in rat coccygeal discs; 14 days after injection a decrease in the number of MSC labeled with fluorescence was observed.However, twenty-eight days after injection the number of MSC rose to the initial number of cells with 100% viability.
Bendtsen et al. 36 induced disc degeneration in minipigs and injected hydrogels without MSC and hydrogels loaded with autologous MSC.They found that MSC and hydrogel therapy are able to partly regenerate the IVD and maintain perfusion and permeability of the vertebral endplate and subchondral bone.Another author performed the transplantation of human MSC into minipigs with DDD and found that those cells survived in the porcine disc for at least 6 months and expressed typical chondrocyte markers suggesting differentiation toward disc-like cells, demonstrating the possibility of xenotransplantation 37 .Some authors also reported the increase in disc height with the use of MSC compared to control groups [38][39][40] .
The in vivo studies performed in rats and minipigs were able to demonstrate that MSC are able to survive and differentiate when injected into the degenerated IVD and have the potential to restore its normal function and structure.However, those results are limited by its models of degeneration that trigger rapid degeneration of the IVD which are not equivalent to slow degeneration that occurs in human disc degeneration 41 .Also, the tail disc of rats and pigs is not submitted to the same static and dynamic load to which human discs are exposed 42,43 .

Clinical Studies with Stem Cells and Disc Degeneration
There are a few clinical studies using stem cells in DDD in the literature.
Haufe and Mork 7 found no improvement in the lower back after one year in 10 patients with DDD submitted to injection of hematopoietic precursor stem cells obtained from the patient's bone marrow.However, those authors did not perform any culture or expansion of the stem cells prior to the injection.In addition, they also submitted the patients to a 2-week course of hyperbaric oxygen therapy after the cells were injected.
Yoshikawa et al. 9 studied the effect of MSC in two patients with DDD; they found that two years after the initial procedure the symptoms had improved in both patients and the rates of the Visual Analog Scale score for low back pain have decreased 38% in one case and 18% in the second patient.In both patients after two years radiograph and computed tomography confirmed that the intervertebral vacuum phenomenon improved.
Orozco et al. 8 performed injected autologous MSC in 10 patients with confirmed DDD; they found that after the injection of the MSC the patient had a rapid improvement in pain and disability at 3 months, followed by a modest improvement within 6 and 12 months after injection.There appeared to be no improvement in disc height.However the water content of the disc was significantly elevated at 12 months.This author also pointed out that those results are important, since intervention is simpler, more conservative, preserves normal biomechanics, and does not require surgery or patient hospitalization 8 .

finaL cOnsiDeRaTiOns
IVD degeneration is a natural process throughout human life and it involves many changes in the composition of the disc structure.Reduction of cellularity and the consequent changes in extracellular matrix are key factors in the physiopathology of the degenerative process.The use of cell therapy for IVD degeneration has been studied as a potential treatment option for this condition.This therapy is supposed to allow treatment of patients with back pain, avoiding extensive surgeries, with less expense, besides leading to less morbidity.The use of MSC is being widely studied and shows a great potential in the repopulation on the NP.Although many advances have been made and many experimental models have shown that MSC are able to regenerate the disc, so far there are few clinical studies measuring the effect of those cells in humans with degenerative disc disease.