OMNIHOSPITAL - SOLCA – UCSG
Cirugía Traumatológica Ortopédica y Medicina Celular
Universidad de Maastrich
Ensayo Clinico, Randomizado, No Ciego: Tratamiento Regenerativo con Celulas Madre Autologas Mesenquimales en la Osteoporosis de Rodilla.
–TRECEMAM – (nombre del estudio)
- Dr. Carlos Chiriboga Accini.
- Dr. Mario Murgueitio Eguez.
- Dra. Aurora Romero Coronel
- Dr. Peter Chedraui Alvarez
- Dr. Danilo Gavilanes Jiménez.
- Dr. Iván Cherrez Ojeda.
- Dr. Santiago Trujillo.
ESTUDIO EXPERIMENTAL DE TIPO ENSAYO CLINICO, RANDOMIZADO, NO CIEGO, DE TRATAMIENTO REGENERATIVO CON CELULAS MADRE AUTOLOGAS MESENQUIMALES EN LA OSTEOARTROSIS DE RODILLA.
–TRECEMAM – (nombre del estudio)
Dr. Carlos Chiriboga Accini:
Jefe de la Unidad de Docencia, Asistencia e Investigación Omnihospital.
Miembro del Staff de Traumatología y Ortopedia Omnihospital.
Coordinador del Posgrado de Ortopedia y Traumatología de la UCSG
Director del Centro de Ortopedia y Control del Dolor. Omnihospital
Dr. Mario Murgueitio Eguez:
Profesor de postgrado Pediatría de la Universidad Católica de Guayaquil.
Ex Jefe de Servicio de Ortopedia y Traumatología del Hospital Francisco Icaza
Jefe de Servicio del Staff de Traumatología y Ortopedia Omnihospital.
Dra. Aurora Romero Coronel
Jefe de Biología Molecular y Citometría de Flujo.
Hospital de SOLCA.
Dr. Peter Chedraui Alvarez
Director del Instituto de Biomedicina
Universidad Católica de Santiago de Guayaquil
Dr. Danilo Gavilanes Jiménez.
Profesor Investigador del Instituto de biomedicina UCSG.
Profesor Adscrito de la Universidad de Maastrich Holanda.
Dr. Iván Cherrez Ojeda.
Magister en Investigación y Epidemiología Clínica.
Director del Centro de Investigación RespiraLab.
Dr. Santiago Trujillo.
Médico residente Omnihospital
Las células madre mesénquimales (MSC) son células primitivas que tienen la capacidad de replicarse y diferenciarse en múltiples líneas celulares, como son los osteocitos, condrocitos, adipocitos entre otros. Las MSC adultas y autólogas pueden ser aisladas de varios tipos de tejidos como pueden ser el cordón umbilical, la médula ósea y el tejido adiposo. Las propiedades multipotentes regenerativas de las MSC las convierten en una opción prometedora en el tratamiento de la Osteoartritis (OA). Los tratamientos actuales de la OA están dirigidos a disminuir el dolor e inflamación pero no modifican el curso de la enfermedad. Los casos más avanzados de OA requieren reemplazos articulares metálicos, con altos costos al sistema de salud nacional. El objetivo de este estudio es comparar tres tipos de tratamientos en la OA de rodilla con MSC obtenidas de dos tipos diferentes de tejidos, MSC de médula ósea versus MSC de tejido adiposo, versus MSC combinado de médula ósea y tejido adiposo en un ensayo clínico randomizado no ciego. Existen centenares de estudios publicados sobre MSC en Osteoartritis de rodilla, pero ninguno compara los resultados con ambos tipos de MSC de diferente origen de extracción. El resultado que podría tener este tipo de tratamiento en la economía de nuestro sistema de salud puede ser altamente significativo bajo un programa de tratamiento correcto y con un entrenamiento adecuado a los profesionales de la salud que trabajan con esta enfermedad.
El propósito de este estudio es evaluar la eficacia que tiene el tratamiento regenerativo con células madre autólogas adultas mesénquimales provenientes de médula ósea (BMSC: Bone Marrow Stem Cells) versus el tratamiento con células madre mesénquimales provenientes de tejido adiposo (AD MSC: Adipose Tissue Derived Mesenquimal Stem Cell) y el tratamiento con la combinación de ambos tipos BMSC Y ADMSC en los pacientes con OA de rodilla grados I, II y III.
OMNIHOSPITAL - SOLCA – UCSG UNIVERSIDAD DE MAASTRICH
PUBLICACIONES EN DIVERSOS MEDIOS.
Bone marrow versus adipose mesenchymal stem cells in osteoarthritis knee treatment. A Randomized no blind controlled clinical trial.
Carlos Chiriboga A., Mario Murgueitio E., Iván Cherrez O., Juan Carlos Calderón. , Aurora Romero C., Danilo Gavilanes., Peter Chedraui.
- Universidad Católica Santiago de Guayaquil.
- Respiralab Research Center
- Sociedad de Lucha contra el Cáncer. SOLCA
Mesenchymal stem cells (MSCs) are stromal cells that have the ability to self-renew and also exhibit multilineage differentiation. MSCs can be isolated from a variety of tissues, such as umbilical cord, bone marrow, and adipose tissue. The multipotent properties of MSCs make them a promising option to treat osteoarthritis (OA).
Bone marrow Mesenchymal stem cells (BM-MSC) and Adipose derived Mesenchymal stem cells (AD-MSC) have been used separately to treat OA. The aim of the present study will be to compare three kind of intra-articular injections of MSC populations obtained from two clinically relevant sources: injections containing BM-MSC or AD-MSC or both. In a randomized no blind clinical trial.
Mesenchymal stem cells (MSC) represent an archetype of multipotent somatic stem cells that hold promise for application in regenerative medicine 1 . During the last decade there is an intensive investigation and increasing number of reports on the treatment of OA using MSC.2,3,4
Osteoarthritis is the most common degenerative joint disease, involving progressive degeneration of the articular cartilage and sub-chondral bone along with synovitis5. Cartilage degeneration may occur in response to inappropriate mechanical stress and low-grade systemic inflammation associated with trauma, obesity, and genetic predisposition, which are major risk factors of OA development and progression 6,7
Current treatments options for OA are aimed to relieve inflammation and pain, but have no effect on the natural progession of the disease 8. Despite many treatments available, in many cases, surgically substitution with metallic implants is inevitable. In 2013, 930,000 hip and knee joint replacements were performed in the United States. 16.6% of them were subject to septic and aseptic surgical revisions, resulting in millions of dollars of costs for the health care system9.
There are many surgical treatments options to repair an articular cartilage defect including abrasion chondroplasty, subcondral drilling, microfracture, mosaicoplasty and more techniques. These procedures are, however, limited to the repair of focal defects and consequently we lack a reparative technique for the more global/diffuse pathology of OA.
Knee OA is not just a articular cartilage defect, it involves the entire joint including subchondral bone thickening, osteophyt formation, synovial inflammation, and degeneration of ligaments and menisci10. OA is a multifactorial disease that involves alterations in cellular and metabolic activities, resulting in tissue degeneration11.
To this date, no drugs are available to structurally modify OA processes or prevent progression of the disease12.. The use of mesenchymal stem cells (MSCs) as a treatment option in cartilage regenerative therapies is under extensive investigation.
Whilst evidence of the capacity of MSCs to differentiate along a chosen cell lineage represents great promise in the area of regenerative medicine it is postulated that their beneficial effect is also achieved through an immunomodulatory and paracrine mechanism and hence manipulation of the disease process13.
In an inflammatory environment, MSCs secrete factors which cause multiple anti-inflammatory effects and influence matrix turnover in synovium and cartilage explants. . The whole panel of bioactive factors probably works in concert to achieve the anti-osteoarthritic effects observed14.
Stem cells have an important role in the maintenance and regeneration of tissues and they are located in a specific microenvironment, defined as niche23. Extracellular matrix (ECM) or micro cellular environment or “Niche” has a fundamental role in cellular biology regulating by direct or indirect action cellular behavior. The ECM is a dynamic and versatile compartment modulating the production, degradation and remodeling of its components, thus giving support for the development, function and repair of tissues23.
The key role of ECM in regulating cell behavior now represents a well-established fact and this concept is especially critical for stem cells, which are defined by a unique and specialized niche in which ECM represents an essential player.23 For these reasons we prepare all the patient before MSC procedures, regulating the ECM (Materials & methods)
The use of intrarticular injections of mesenchymal stem cells (MSC) in combination with PRP (Platelet Rich Plasma) may represent a treatment of the “whole joint”. Along with their immunomodulatory and differentiation potential, MSCs have been shown to express essential cytokines including Transforming Growth Factor beta (TGFβ), Vascular Endothelial Growth Factor (VEGF), Epidermal Growth Factor (EGF) and an array of bioactive molecules that stimulate local tissue repair15, 16. These trophic factors, and the direct cell to cell contact between MSCs and chondrocytes, have been observed to influence chondrogenic differentiation and cartilage matrix formation17,18.
Recent data indicate that these paracrine factors, because their inmuno modulation and differentiation potential, may play a key role in MSC-mediated effects in modulating various acute and chronic pathological conditions22.
Like mesenchymal stem cells from bone marrow (BM-MSC), adipose tissue-derived adult stem cells (AD-MSC cells) can differentiate into several lineages and present therapeutical potential for repairing damaged tissues19, 20.
The standard site for obtaining human MSC is bone marrow, however, one limitation for obtaining MSC from bone marrow is the difficulty of obtaining enough number of cells required for clinical studies. Adipose tissue can be obtained by less invasive methods and in larger quantities than bone marrow cells, making the use of AD-MSC as a source of stem cells very appealing21. AD-MSC seem to be as effective as BM-SC in clinical application, and, in some cases, may be better suited than BM-MSC22.
The aim of this study is to analyze in 3 groups of patients with knee OA grade II and III, and the capacity of MSC to delays or even reverts the cartilage damage of OA.
A phase III prospective, triple arm, open label dose would be conducted of a single injection of BM-MSC; AD-MSC; and BM-MSC+AD-MSC in 3 groups of patients with knee OA grade II and III (Ahlback classification) diagnosis. This study will be conducted from January 2018 to June 2019 in Omnihospital.
Group 1 with 12 patients is going to receive BM-MSC. Group 2 with 12 patients will receive AD-MSC. And a 3rd group will receive BM-MSC combined with AD-MSC. All 3 groups are going to receive PRP (Platelet Rich Plasma) at the time of the procedure.
All 3 groups of patient are preparing before the procedure acting on ECM through procaine injections in order to repolarize cell membrane of the areas to work. It improves the metabolic processes, including cellular respiration. Procaine has a high redox potential of + 290 mV. It shows a protective effect on the membrane’s cells, stimulates cellular regeneration and the regeneration of the basic substance.
Flow citometry is going to be used in all patients, in order to know the amount and types of MSC injected. X ray and magnetic resonance with T2 Map imaging will be performed to analyse cartilage damage. The 6 months control appointment includes new T2 Map MRI. Pain and function will be assessed using VAS and WOMAC scales.
- A randomized no blind clinical trial with active control. Patients with knee osteoarthritis grade II and III diagnosis acording Ahlback classification(22)would be completed the selection criterious.
- All patients must complete an informed consent.
- Varus or valgus knee mal alignment superior to 15°.
- OA grade IV according Ahlbäck classification because mechanical instability..
- Bone marrow cáncer like lymphoma. .
- Severe anemia.
- Active infections.
- Clinical and orthopedic evaluation.
- Complete blood test..
- X Rays and T2 Map MRI
- Extracelular Matrix (ECM) preparation.
- Infiltration with procaine 2% by repolarization of the cell membrane improves ECM at the areas to work.
- The procedures will be performed in the operating rooms of the hospital.
- Ambulatory surgery. Type of Anesthesia: local anesthesia and sedation.
- Average procedure time: 75 minutes.
- Bone marrow aspiration . Standard surgical technique: Percutaneous puncture of posterior iliac crest Fluoroscopy guided.
- Aspirate 60 to 80 ml of blood from bone marrow.
- Place the aspirate in green (heparin) tubes and centrifuge at 2800 rpm for 10 minutes.
- Aspirate the upper clot of the tubes, obtaining the supernatant.
- Place 1 ml in purple cap tube for flow cytometry test.
- Apply the percutaneous injection of bone marrow aspirate into the affected knee.
- Apply local anesthesia with Klein tumescent solution in the region of greater trochanter of hip and gluteus.
- With 60 ml syringe and 2.5 to 3 mm diameter cannula extract 30 ml of pure fat.
- Transfer the 30 ml of fat to 2 syringes of with equal luer lock spike.
- Add physiological solution and decant discarding infranadante to remove traces of tumescence and blood. (Repeat 2-3 times).
- Sterile introduction of 4 conical tubes of 50 ml, collagenase bottle, 50 ml syringe, 18 g needle and 0.22 micron-
- Place the washed adipose tissue in 4 conical tubes of 50 ml at a rate of 15 ml per tube.
- Reconstitute the collagenase using a 60 cc syringe with saline solution. Connect the 0.22 micron filter to the syringe and add 15 ml of the reconstituted collagenase to each tube.
- Shake the tubes for 20 minutes by placing in the heater block or shaker. Centrifuge the two tubes at 900g for 5 minutes.
- Carefully remove the fat and saline supernatant until the pellet leaves approximately 5 ml. Using the pipette connected to the 20 ml syringe extract the pellet of Stromal vascular factor.
- Transfer the obtained pelllet in each tube to another sterile 50 ml tube. Add to each tube physiological solution until the 45 ml is completed.
- Aspirate again with a 20 ml syringe and pipette the button of each of the tubes, taking off the cells of the conical bottom. A total volume of about 5 to10 ml is obtained.
- Place cell strainer in a sterile 50 ml tube, gently transfer the cell suspension through the strainer.
- 1 cc of the adipose tissue stem cell concentrate is sent to the MOLECULAR BIOLOGY laboratory for quantification by flow cytometry of the volume and viability of obtained stem cells.
- Apply the percutaneous injection of ADMSC into the affected knee.
- The two previous procedures are repeated and Mesenchymal stem cells of fatty tissue are joined with bone marrow stem cells.
- Intraticular percutaneous knee injection of the AD MSC + BMSC is started immediately after their extraction and preparation.
- 1 ml of the adipose tissue stem cell concentrate is sent to the MOLECULAR BIOLOGY laboratory for quantification by flow cytometry of the volume and viability of obtained stem cells.
The present investigation is a PILOT STUDY.
We used G*Power Version 3.0.10 to determine the sample size. We applied the parameters for the selected F test, two-way mixed ANOVA (repeated measures, within-between interaction). Given the fact that no technique has been previously compared altogether, we performed the calculation considering a medium size effect (f=0.25). We set the α error probability to 0.05 and a power of 0.80 (actual power 0.82). We calculated applying 3 repetitions, though we will perform 6 measurements for some variables (for instance, VAS or WOMAC questionnaire). Applying these criteria, the total sample size would be 36, meaning each group will need 12 individuals. For more details, we provide the output below.
H1: The combined use of BM-MSC and AD-MSC is superior for pain management in knee OA, than the use of BMSC alone or ADMSC alone. Its application in the OA protocols can change the course of the disease and reduce the costs of it.
H0: The combined use of mesenchymal stem cells from bone marrow and adipose tissue is not superior to the use of stem cells derived solely from bone marrow or from adipose tissue alone in the treatment of pain and function in patients with knee OA.
This study will apply descriptive statistics to determine frequency and proportions for variables such as gender, ethnicity and knee osteoarthritis severity, as well as mean and SD for age, WOMAC score (and each component of the scale) and VAS.
We will apply a two-way mixed ANOVA test to compare the mean differences between treatment groups for each dependent variable, i.e. WOMAC score, VAS score, radiologic changes and health expenditure. With respect to WOMAC score, we are not only going to analyze the overall score, but also each of its three components separately (pain, stiffness and function). The tests will determine if there is an interaction between these variables and the factors (independent variables). The factors for our analysis are type of treatment (between-subjects factor) and time (within-subjects factor). The treatment categories are BMSC alone, ADSC alone and BMSC + ADSC. Concerning to the factor time, it will differ according to what dependent variable is subject to analysis. For instance, WOMACK score and VAS score analyses are going to be evaluated in 6 different time frames (patients will be asked to answer the questionnaires/scales 3 weeks before intervention and for the first, second, third, sixth, twelfth and twenty-fourth month after surgery). For radiologic changes, T2 mapping MRI is going to be performed only twice to reduce the patient’s exposure to radiation, that is before the procedure and 6 months after. Health expenditure will be quantified 2 years before intervention and 2 years after the procedure.
To ensure the reliability of the analyses, we will test for outliers applying boxplots, and test for normality using the Shapiro-Wilk method. Furthermore, homogeneity of variances will be assessed by Levene’s test, while homogeneity of covariances will be explored by Box’s M test. Then, to meet the assumption of sphericity, Mauchly’s test will be performed. In case of assumption violation, other tests will be carried out to analyze the data.
Finally, generalized linear mixed models are going to be performed to determine how likely are each of the interventions to present complications. The complications considered in this work will be pain and hematomas at the surgical site where stem cells where extracted, synovial effusion and knee pain.
All the data will be analyzed using SPSS, version 24.0 software (SPSS Inc., Chicago, IL, USA). A p-value of less than 0.05 will be considered statistically significant.
Absence of immunological reaction and disease transmission because it belongs to the same patient. Wakitani24 from the University of Osaka Japan demonstrated in an 11-year follow-up study that MSC did not induce tumor growth or infection in any patient treated for cartilage lesions.
Chris and colleagues25 report the use of intra-articular injection of 1.0 × 108 AD MSCs in patients with knee OA with results of improvement in joint function without causing adverse events.
Peetersy et al26. evaluated 8 studies involving 844 MSC-treated patients with intra-articular injected culture expansion. , they conclude that there are no opposing arguments for the intra-articular application of MSC.
Systematic reviews have all been in favor of the safety in intraarticular – injections of MSCs. However, great caution is warranted with culturing and expansion of MSCs27.
PLATELET-RICH PLASMA (PRP)
The function of MSCs has been explored under the influence of bioactive carriers such as platelet-rich plasma (PRP). Platelets contain greater than 1500 protein based factors with bioactive ability32. This broad spectrum of compounds includes growth factors, peptide hormones, chemokines, fibrin and also proteins with anti-bacterial and fungicidal properties.
Growth factors released by platelets may potentially play a positive role in the up regulation of MSCs. TGFβ1 is seen to reduce collagen type I gene expression and up regulate expression of collage type II and aggrecan genes33. Further, TGFβ1 works in association with basic Fibroblast Growth Factor (FGF2) to assist in the migration of stromal cells to a site of injury35,36.
The combination of PRP with MSCs in intra-articular injections has shown increased collagen type II expression and reduced chondrocyte apoptosis34.
ADIPOSE DERIVED MSC
Most recently, Phase I and II trials using expanded adipose derived MSCs in the treatment of OA have shown MRI evidence of cartilage regrowth31. Following a single intra-articular injection of 100 million MSCs, radiological (MRI) follow-up at 6 months showed increased cartilage volume.
Using a combination of both isolated bone marrow MSCs, BMA and platelet lysate, Centeno and colleagues have published the observed improvement in both chondral volume and meniscus volume in two limited case studies28,29. In 2011, Centeno later published a case series of 339 patients, reporting that of those patients requiring total knee replacement (69 % of the patient cohort) only 6.9 % still required replacement surgery after MSC therapy. Sixty percent of patients reported >50 % pain relief and 40 % reported >75 % pain relief at 11 months29.
A recent Phase 1 dosing trial on the use of adipose derived MSCs in severe osteoarthritis indicated a significant effect over a 12 month follow-up on the need for total joint replacement with only 2 out of the 18 patients still requiring arthroplasty37. This is similar to Centeno’s observation of the effect of MSC based therapy in delaying need for joint replacement.
The success of such combination therapy has also been indicated by a limited case series assessing the benefits of adipose derived MSC, where MSC was combined with both a platelet lysate and a hyaluronic acid carrier with additional use of low dose dexamethasone30. Again, both functional and disease modification was observed.
Osteoarthritis is a leading cause of pain and disability. With an aging population its prevalence is even going to increase. Current standard treatments target symptomatic relief rather than the underlying mechanisms and hence prevention. Medical treatments do not change the natural course of this disease and involve the use of drugs with a high percentage of complications. Often conducted surgical interventions are accompanied by significant risks. More advanced cases require very costly surgeries for health systems, and they also have many serious complications.
Encouraging results with MSC from pre-clinical and clinical trials have provided initial evidence of safety and efficacy for many age-, inflammation- or degenerative-related diseases, including osteoarthritis (OA). Systematic reviews have all been in favor of the safety in intraarticular – injections of MSCs.
Numerous clinical trials have been published on the use of MSC in osteoarthritis. None of them compare the results between the use of BM MSC origin and AD MSC, and the combination of both types. All of them adding Platelet Rich Plasma to increase the chondrogenic differentiation and cartilage matrix formation.17,18
The intervention is simple, does not require hospitalization or open surgery, provides pain relief, would significantly improves cartilage quality, could change natural progression of the disease and improve quality of life of these patients.
In the future treatments with MSC may become the treatment of choice in knee osteoarthritis.
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- Wakitani S., Imoto K., Yamamoto T., Saito M. Human Autologous culture expanded bone marrow mesenchymal cell transplantation for repair of cartilage defects in osteoarthritic knees. Osteoarthritis and Cartilage, Volumen 20, Issue10. October 2012. Pages 1186–1196.
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