RE: Disc Regeneration Therapy using Marrow Mesenchymal Cell Transplantation: A Report of Two Case Studies

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Perhaps this office can help find Drs.here in Oregonthat use stem cells for lumbar disc regeneration? : http://www.regenexx.com/about-regenexx/doctor-christopher-j-centeno-md/ RegenerativeSciences403 Summit Blvd., Suite 201Broomfield, CO 80021www.regenexx.comPhone:303-495-4014Toll Free: 1-888-525-3005s. fuchs dc -----OriginalMessage-----From: Sharron Fuchs <sharronf@...>Oregon DC < >Sent: Mon, Jun 28, 2010 11:51 amSubject: Disc Regeneration Therapy using Marrow MesenchymalCell Transplantation: A Report of Two Case Studies From Spine Disc Regeneration Therapy using Marrow Mesenchymal CellTransplantation: A Report of Two Case StudiesTakafumi Yoshikawa, MD; Yurito Ueda, MD;Kiyoshi Miyazaki, MD; Munehisa Koizumi, MD; Yoshinori Takakura, MDPosted: 06/11/2010; Spine. 2010;35(11):E475-E480. © 2010 Lippincott & WilkinsAbstract and IntroductionAbstractStudy Design. Marrow mesenchymal cells (MSCs) contain stem cells and possessthe ability to regenerate bone, cartilage, and fibrous tissues. Here, weapplied this regenerative ability to intervertebral disc regeneration therapyin an attempt to develop a new spinal surgery technique.Objective. We analyzed theregenerative restoration ability of autologous MSCs in the markedly degeneratedintervertebral discs.Summary of Background Data. Fusionfor lumbar intervertebral disc instability improves lumbago. However, fusedintervertebral discs lack the natural and physiologic functions ofintervertebral discs. If intervertebral discs can be regenerated and repaired,then damage to adjacent intervertebral discs can be avoided. We verified theregenerative ability of MSCs by animal studies, and for the first time,performed therapeutic intervertebral disc regeneration therapy in patients andobtained favorable findings.Methods. Subjects were 2 womenaged 70 and 67 years; both patients had lumbago, leg pain, and numbness.Myelography and magnetic resonance imaging showed lumbar spinal canal stenosis,and radiograph confirmed the vacuum phenomenon with instability. From the iliumof each patient, marrow fluid was collected, and MSCs were cultured using themedium containing autogenous serum. In surgery, fenestration was performed onthe stenosed spinal canal and then pieces of collagen sponge containingautologous MSCs were grafted percutaneously to degenerated intervertebraldiscs.Results. At 2 years after surgery,radiograph and computed tomography showed improvements in the vacuum phenomenonin both patients. On T2-weighted magnetic resonance imaging, signal intensityof intervertebral discs with cell grafts was high, thus indicating highmoisture contents. Roentgenkymography showed that lumbar disc instabilityimproved. Symptom was alleviated in both patients.Conclusion. The intervertebraldisc regeneration therapy using MSC brought about favorable results in these 2cases. It seems to be a promising minimally invasive treatment.IntroductionLumbar spine instability causes low backpain, and in intervertebral disc instability, dynamic damage to nerve rootsinduces lower extremity neurologic symptoms. In patients who do not respond toconservative therapy, spinal cord decompression and spinal fusion have beenperformed. Spinal fusion stabilizes the lumbar spine, thus improving low backpain, and because nerve roots are not dynamically damaged in fusedintervertebral spaces, neurologic symptoms also improve.[1–4] However, spinal fusion eliminates thenatural and physiologic functions of intervertebral discs. Intervertebral discsconnect the spine, contain nucleus pulposus, and are surrounded by anulusfibrosus. Nucleus pulposus is a highly elastic gel, and it absorbs shockapplied to the spine. Spinal fusion places more stress on those discs adjacentto fused intervertebral discs, thus accelerating the regressive degeneration ofadjacent discs. In the long term, prognosis for fusion can be poor.[5–8]Also, it has problems with damage because of autologous bone harvesting forfusion.[9–14] The ideal treatment is to conserve thephysiologic function of intervertebral discs and flexibility of the spinewithout spinal fusion. If it were possible to regenerate intervertebral discs,the prognosis for spinal surgery would improve markedly. Although research hasbeen conducted on intervertebral disc regeneration using somatic stem cells,[15–23]to the best of our knowledge, there have been no reports on intervertebral discregeneration therapy in clinical settings.We previously reported that intervertebraldisc-like tissue could be regenerated by grafting cultured marrow cells todamaged intervertebral discs, and that the tissue-regenerating ability ofmarrow cells could be applied to intervertebral disc regeneration.[20]About 3 years ago, we were the first group to perform intervertebral discregeneration therapy using marrow cells in 2 patients, and favorable resultswere obtained.Materials and MethodsThe present disc regeneration therapyusing marrow mesenchymal cells (MSCs) was submitted to the university's EthicsReview Board, was approved in June 2004, and was implemented in March 2005. Thepresent therapy is the first of its kind to be performed anywhere in the world,and the following explanations were given to the patients and their familieswhen obtaining written informed consent: the objectives and methods of thepresent therapy; expected effects and risks; other possible treatments; refusalto consent to participate in this study would not result in any negativeconsequences; consent could be withdrawn at any point; and privacy would beproperly protected. Therapy was performed after obtaining informed consent.Subjects satisfied the following criteria:(1) magnetic resonance imaging (MRI) confirmed intervertebral discdegeneration; (2) radiograph showed the vacuum phenomenon; (3)roentgenkymography revealed intervertebral disc instability; (4) at the levelof the degenerated intervertebral disc associated with the vacuum phenomenonand instability, pressure and spontaneous pain were seen, and when wearing acorset, low back pain was alleviated, and thus, the patients were diagnosedwith symptomatic disc; and (5) complicated by lumbar spinal stenosis andneurologic symptoms, resisted conservative therapy, and required surgery.Both the patients were women, andradiograph confirmed marked intervertebral disc regressive degeneration and thevacuum phenomenon. Patients had intervertebral instability, low back pain, andlower extremity neurologic symptoms, and they did not respond to conservativetherapy. The vacuum phenomenon refers to intervertebral regressive degenerationand cavitation that appears as dark spots on radiographs. The clinicalsignificance of tissue regeneration in intervertebral cavities is substantial.Intervertebral disc regeneration improves the vacuum phenomenon, facilitatingassessment of postoperative intervertebral disc regeneration.Marrow Mesenchymal Cell Culturing and GraftingPreparation of HumanMarrow Mesenchymal Cells. Approximately 5 mL ofbone marrow fluid was aspirated from the ilium under local anesthesia. Toprevent bone marrow fluid coagulation, 1 mL heparin was placed in a 10-mLinjector beforehand (1000 U, Fuji Pharma Co., Japan) and was mixed with the bonemarrow fluid. Bone marrow fluid was centrifuged (1000 rpm for 5 minutes), andafter eliminating the supernatant and adding 5 mL physiologic saline, it wasagain centrifuged. The supernatant was aspirated, and the heparin was removedbefore incubation. Bone marrow fluid was placed in a T75 flask (Falcon) andplaced in a carbon dioxide incubator (temperature, 37°C; humidity, 100%; and CO2,5%). Eagle minimum essential medium (Sigma Co., St. Louis, MO) containing 15%autologous serum, an antibiotic (gentamicin; Schering Plough), and 100 nmol/Lestriol (Nacalai Tesque, Kyoto, Japan) was used.[24] Culture mediumwas exchanged 3 times per week. After 2 to 4 weeks (4 weeks in case 1 and 2weeks in case 2), cultured cells were treated with 0.1% trypsin (NacalaiTesque), and the resulting cells were centrifuged at 1000 rpm for 5 minutes.Centrifuged cells were rinsed once using physiologic saline, and a cellsuspension of 105 cells/mL was prepared. Before surgery, culturemedium was tested for bacteria, fungi, mycoplasma, and endotoxins to confirmthe absence of microbial infection.Cultured cells were placed in a 10-mLdisposable injector, packaged aseptically, and brought to the operating room inan ice chest. About 20 pieces of collagen sponge were placed in the autologousMSC suspension for about 10 minutes to ensure cell penetration. In cellgrafting, the instruments for percutaneous nucleus pulposus removal were used,and under radiograph guidance, a narrow tube was inserted into a targetintervertebral space to graft pieces of collagen sponge containing autologousMSCs 1 at a time, and the cells were exactly grafted into the central regionsof discs as shown in Figure 1. After grafting of the cells, the hole was closedwith collagen sponge (without cells).Figure 1. Marrow cells were cultured for 2 to 4 weeks. Pieces ofcollagen sponge were soaked in cultured mesenchymal cell solution andpercutaneously grafted to intervertebral disc cavities under radiographicguidance.The collagen sponge was prepared from theartificial dermis (Pelnac; Gunze Limited Co., Japan[25]) whichconsists of a 150-μm-thick silicone film and a 3-mm-thick collagen sponge witha gap size of 70 to 100 μm and porosity of 80% to 96%. The collagen sponge wascut into 3 mm × 3 mm squares, the silicone membrane was removed, and 1 mL ofthe above cell suspension was soaked onto it. About 20 pieces of the collagensponge were grafted into the degenerated disc.Clinical Course for Case 1The patient was a 70-year-old woman havinglow back pain and right lower leg pain as chief complaints. About 15 yearspreviously, the patient underwent anterior interbody fusion of L4–L5 because ofleft lower leg numbness and low back pain. At about 6 years after surgery, shebegan to experience low back pain and right lower leg numbness, and because hersymptoms did not improve, she visited the hospital. Radiograph showed favorableanterior interbody fusion and spinal decompression in L4–L5, but intervertebralvacuum phenomenon, instability, and lumbar spinal stenosis were observed inL2–L3 and L3–L4 (left images in Figure 2). Because of anterior interbody fusionin L4–L5, we determined that fusion of the superior lumbar vertebrae would beinappropriate, and as a result, intervertebral disc regeneration therapy wasrecommended, and the patient consented.Figure 2. Radiograph and MRI images before (left column) and 2 yearsafter (right column) surgery in case 1. A preoperative myelogram (upper left)showed lumbar spinal stenosis in L2–L3 and L3–L4 (arrow) and intervertebralvacuum phenomenon. A radiograph taken at 2 years after surgery showed that theintervertebral vacuum phenomenon in L2–L3 and L3–L4 (arrow) intervertebral dischad improved. T2-weighted MRI showed that the signal intensity in L2–L3 andL3–L4 (arrow) was low before surgery (lower left), but at 2 years aftersurgery, the signal intensity in L2–L3 and L3–L4 (arrow) was high, thusindicating increased moisture content.Five milliliters of marrow fluid and 100mL blood for autologous culture serum preparation were collected. AutologousMSCs were prepared as described earlier. After culturing for 4 weeks andconfirming cell proliferation, fenestration was performed on L2–L3 and L3–L4.Pieces of collagen sponge were then soaked in the MSC solution, and underradiograph guidance, cells were grafted into the central regions of both discspercutaneously as shown in Figure 1.Postoperative radiograph showed thatintervertebral disc cavities were filled with collagen sponge pieces containingMSCs, and after surgery, vacuum phenomenon improved. For 2 weeks after surgery,the patient stayed in bed to allow the surgical wounds to heal, and using aflexible corset, standing and walking rehabilitation was initiated. Symptomsimproved, and the patient was discharged at 1 month after surgery. The patientwore the flexible corset for 2 months after surgery.At 6 months after surgery, low back paindisappeared, and left lower leg numbness and pain was alleviated. The patientthen experienced mild low back pain, but at 2 years after surgery, the VisualAnalog Scale score for low back pain has decreased to 38%, and symptoms haveimproved. Japanese Orthopaedic Association scores also improved from −3 to 9points.At 2 years after surgery, radiographsconfirmed that the vacuum phenomenon in the intervertebral disc spaces hasimproved (Figure 2). Although no notable findings were seen on T1-weighted MRI,signal intensity of the intervertebral discs with cell grafts was higher onT2-weighted images, indicating high moisture content (lower images in Figure2).Clinical Course for Case 2The patient was a 67-year-old woman.Because low back pain and leg pain persisted for several years, the patient wasreferred to the hospital to undergo surgery. Plain radiograph showed L4–L5instability and vacuum phenomenon. Myelography showed lumbar spinal stenosis inL4–L5 (left image in Figure 3 and left image in Figure 4). Marrow fluid andblood for culture serum preparation were collected, and autologous mesenchymalcells were cultured as described earlier. Culture was performed for 2 weeks,and cell proliferation was confirmed.Figure 3. Preoperative myelography and computed tomography (CT)(left), radiograph taken 2 years after surgery (middle) and CT taken 2 yearsafter surgery (right) in case 2. Preoperative myelography (left) confirmedL4–L5 vacuum phenomenon and lumbar spinal stenosis. Radiograph (middle) and CT(right) taken 2 years after surgery showed that L4–L5 vacuum phenomenon hadimproved.Figure 4. MRI images before (left) and after (right) surgery in case2. On T2-weighted MRI (lower), the signal intensity of L4–L5 intervertebraldisc was low (lower left), but at 2 years after surgery (lower right), thesignal intensity of L4–L5 intervertebral disc was high, indicating increasedmoisture content.After performing fenestration in L4–L5, asin case 1, pieces of collagen sponge containing autologous MSCs were graftedpercutaneously in L4–L5 (Figure 1). After 2 weeks of bed rest after surgery,standing and walking rehabilitation was initiated using a flexible corset.Symptoms improved, and the patient was discharged at 1 month after surgery. Thepatient wore the flexible corset for 2 months after surgery.At 2 years after surgery, radiograph andcomputed tomography confirmed that the intervertebral vacuum phenomenonimproved (Figure 3). On T2-weighted MRI, the signal intensity of theintervertebral disc with cell grafts was higher when compared with that beforesurgery, indicating higher moisture content (lower image in Figure 4). Aftersurgery, low back pain, lower leg numbness, and pain improved, and at yearsafter surgery, Visual Analog Scale scores for symptoms decreased to 18%.Japanese Orthopaedic Association scores also improved from 8 to 25 points.DiscussionCase 1 underwent anterior fusion of L4–L5more than 10 years previously, and additional fusion would have been highlyinvasive and would have eliminated the physiologic function of the spine. Inthis patient, the intervertebral disc regeneration therapy that we developedwas suitable for the treatment of intervertebral instability. The affectedintervertebral space in case 2 also exhibited marked degeneration. Severedegeneration was also seen in adjacent intervertebral discs. Withintervertebral disc regeneration therapy, the physiologic function of thelumbar spine was conserved, and low back pain and neurologic symptoms improved.Both the patients were very satisfied with the outcome. At present, research onintervertebral disc regeneration is at the stage of animal studies, but studieshave been conducted on regenerating intervertebral discs using cytokines, genetherapy, autologous intervertebral disc cells, or stem cells.Intervertebral disc degeneration involvescytokines and growth factors.[26,27] Growth factors, such astransforming growth factor-beta, epidermal growth factor, fibroblast growthfactor, and bone morphogenetic protein-7, have been reported to exhibitintervertebral disc repairing effects.[28,29] Although therapy usinggrowth factors is promising, further research on therapeutic effects isrequired. A study on intervertebral disc regeneration therapy based on genetherapy was reported.[30–32] However, the side effects of genetherapy have not been fully clarified, and many ethical issues remain, and as aresult, gene therapy is not yet practical.With regard to research on intervertebraldisc regeneration using stem cells, intervertebral disc regeneration using stemcells that are isolated and extracted from fat tissue seems to be promising.[33]However, a large amount of fat tissue is required for stem cell extraction, andfurther research is required for clinical application. In addition, specializedand expensive equipment is required for stem cell isolation and extraction.Studies have been conducted on the use ofdifferentiated intervertebral disc cells using dog models,[34,35] andthere have been pilot clinical trials. Favorable clinical results were obtainedby collecting intervertebral disc tissue and then isolating, incubating, andproliferating intervertebral disc cells.[36] To obtainintervertebral disc cells, it is necessary to damage healthy intervertebraltissue, collect tissue, and treat it with collagenase for long periods of time.Although differentiated intervertebral cells are practical, proliferationpotential is low, and incubation and proliferation are time consuming.On the other hand, with intervertebraldisc regeneration therapy using MSCs, cells can be obtained by tapping, whichis a minimally invasive procedure, and when compared with separating stem cellsfrom fat tissue or cell separation from intervertebral disc tissue, healthytissue is not damaged for cell collection. This is a convenient technique thatdoes not require specialized equipment or complex procedures, such as long-termcollagenase treatment. There have already been clinical reports on tissue regenerationtherapy using MSCs,[37–43] and such techniques are practical.After performing in vitro studies on intervertebral discregeneration, Yamamoto et al[18] reported that nucleus pulposus cells were activated by cominginto direct contact with MSCs. Risbud et al[19] reported that marrow mesenchymal stem cells differentiated intointervertebral disc cells. In vivo,Crevensten et al [17]grafted rat MSCs to the caudal vertebra and reported that even at 4 weeks aftergrafting, MSCs maintained viability and proliferated within the intervertebraldisc. Sakai et al [16,23]conducted a study on rabbits and reported that grafting mesenchymal stem cellsembedded in atelocollagen gel suppressed intervertebral disc degeneration fromadvancing. Cheung et al [21]conducted a study on rabbits and reported that grafting MSCs to intervertebraldiscs with advanced degeneration was very effective. These results indicatethat grafting MSCs to degenerated intervertebral discs stimulates themetabolism of residual intervertebral disc cells, and as MSCs themselvesdifferentiate into cartilage tissue, intervertebral disc-like tissue isregenerated.We previously performed studies todocument that marrow cells contain stem cells and possess the ability toregenerate various tissues, and investigated their regenerative abilities.[44–51]With approval of the university's Ethics Review Board, we have performedtherapeutic regeneration therapy of various tissues in clinical settings.[39–43]The present intervertebral disc regenerationtherapy using MSC brought about favorable results in these 2 cases. The presenttherapy can be performed through an incision of about 5 mm, and seems to be apromising minimally invasive treatment. With future advances in culturingtechniques and facilities in the field of regenerative medicine, intervertebraldisc regeneration therapy will also advance.SidebarKey PointsIf intervertebraldiscs can be regenerated and repaired, then damage to adjacentintervertebral discs associated with spinal fusion can be avoided, and thelong-term prognosis for spinal surgery would improve.Research onintervertebral disc regeneration by somatic stem cells has been conducted;however, there have been no clinical reports of intervertebral discregeneration therapy.We verified theregenerative ability of marrow mesenchymal cells by animal studies, andfor the first time, performed therapeutic intervertebral disc regenerationin clinical setting.[ CLOSE WINDOW ]ReferencesFoley KT, Holly LT,Schwender JD. Minimally invasive lumbar fusion. Spine 2004;29:598–9.Kim DH, Jaikumar S,Kam AC. Minimally invasive spine instrumentation. Neurosurgery 2002;51(5suppl):S15–25.Pradhan BB, NassarJA, Delamarter RB, et al. 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Bone regeneration by grafting of cultured humanbone. Tissue Eng2004;10:688–98.Yoshikawa T, Iida J,Ueda Y, et al. Bone regeneration by grafting of an autogenous culturedbone/ceramic construct. J Biomed MaterRes A 2003;67:1437–41.No funds were received in support of thiswork. No benefits in any form have been or will be received from a commercialparty related directly or indirectly to the subject of this manuscript.Spine. 2010;35(11):E475-E480. © 2010 Lippincott & WilkinsTakafumi Yoshikawa, MD,* Yurito Ueda, MD,†Kiyoshi Miyazaki, MD,† Munehisa Koizumi, MD,† and Yoshinori Takakura, MD† *Department of Orthopedic Surgery, Koriyama-SeiranHospital, Nara,Japan; and †Department ofOrthopaedic Surgery, Nara Medical University,Nara, Japan.Address correspondence and reprintrequests to Takafumi Yoshikawa, MD, Department of Orthopedic Surgery, Koriyama-SeiranHospital, 1-1 Honjo, Yamatokoriyama, Nara 639-1136, Japan; E-mail: t-yoshikawaseiran (DOT) or.jp s. fuchs dc