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. Emanuel Vanegas, 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 separated to treat OA. The aim of the present study will be to compare three kind of injections of MSC populations obtained from two clinically relevant sources: adipose tissue and the standard bone marrow-derived MSC. And a 3rd group of patients using both, bone marrow and adipose MSC together. 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 availables, 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. Millions of dollars cost the health system by these procedures 9
Articular cartilage defect repair counts with many surgical treatments options, 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 is involved the entire joint organ including the subchondral bone thickening, osteophyte formation, synovial inflammation, and degeneration of ligaments and meniscous10. OA is a multifactorial disease that involves alterations in cellular and metabolic activities, resulting in tissues 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 one essential player.23 For these reasons we prepare all the patient before MSC procedures acting on ECM through procaine injections in order to repolarize cell membrane of the areas to work.
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.
During the last decade there is an intensive investigation and increasing number of reports on the treatment of OA using MSC, The initial results from these trials are very encouraging with different results. In many cases, treatments consist of the use of Bone Marrow MSC (BMSC) as a source of MSC, and other studies use Adipose Tissue to obtain MSC (ADMSC).
Like mesenchymal stem cells from bone marrow (BMSC), adipose tissue-derived adult stem cells (ADMSC 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 ADMSC as a source of stem cells very appealing21. ADMSC seem to be as effective as BMSC in clinical application, and, in some cases, may be better suited than BMSC22.
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.
MATERIALS AND METHODS
A phase III prospective, triple arm, open label dose would be conducted of a single injection of BMSC; ADMSC; and BMSC+ADMSC in 3 groups of 12 patients with knee osteoarthritis grado II and III diagnosis. This study will be conducted from January 2018 to June 2019 in Omnihospital.
Group 1 with 12 patients are going to receive BMSC. Group 2 with 12 patients would receive ADMSC. And a 3rd group would recieve BMSC combined with ADMSC. All 3 groups are going to receive PRP (Platelet Rich Plasma) at the time of the procedure. 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.
Flow citometry is going to be measure in all patients, in order to know the amount and types of MSC injected. X ray and magnetic resonance with T2 Map imaging would be performed to analized cartilage damage. The 6 months control appointmet include new T2 Map MRI. Pain and function are going to be assessed using VAS and WOMAC scales.
- A randomized no blind clinical trial with active control. Patients with knee osteoarthritis grado 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 grado IV according Ahlbäck classification because mechanical instability..
- Bone marrow cáncer like linphoma. .
- Severe Anemia.
- Active Infecctions.
PREPARATION FOR THE PROCEDURE
- 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.
STEM CELL OBTAINING PROCEDURES
The procedures will be performed in the operating rooms of the hospital.
Ambulatory surgery. Type of Anesthesia: local anesthesia and sedation.
Average time procedure: 75 minutes.
BMSC PROCEDURE. GROUP 1 OF PATIENTS.
- Bone marrow aspiration . Standard surgical technique: Percutaneous puncture of posterior iliac crest Fluroscopy 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.
ADMSC PROCEDURE. GROUP 2 OF PATIENTS.
- 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.
BMSC – ADMSC PROCEDURE. GROUP 3 OF PATIENTS.
- 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.
H1: There is a significant interaction between the treatment and time on osteoarthritis management outcomes.
H0: There is no significant interaction between the treatment and time on osteoarthritis management outcomes.
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.
The present investigation is a PILOT STUDY.
Given the fact that no technique has been previously compared altogether, we performed the calculation considering the visual analog scale (VAS) as the parameter. The minimal significant difference is 2.1 to 5.3. We used www.openepi.com, setting a confidence interval of 95%, power of 80% and ratio of sample size of 1. Applying these criteria, the sample size for a mean difference of 2.1 is:
Applying the same criteria, the sample size for a mean difference of 5.3 is:
Thus, in order to demonstrate a statistically significant difference in means of 2.1 we need 15 patients on every group, while if the difference of means is 5.3 it would be required the participation of 28 individuals on each group.
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 and radiologic changes will be measured with T2 maping MRI. 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 maping MRI is going to be done on 6 months 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.
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. We will measure quality of life according to the WOMAC scale. As for costs analysis , it refers to how much the patient has spent on his illness for the last 2 years, and how much the procedure costs
MSC therapy may be a valid alternative treatment for chronic knee osteoarthritis. Current medical treatments do not change the natural course of this disease and involve the use of drugs with a high percentage of complications. More advanced cases require very costly surgeries for health systems, and they also have many serious complications.
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, and would significantly improves cartilage quality.
In the future treatments with MSC may become the treatment of choice in knee osteoarthritis.
- Wagner, Wein Fa, Seckinger A. , Frankhauser M., Wirkner U., Krause U., Blake J., Schwager C., Eckstein V., Ansorge W., Ho A. Comparative characteristics of mesenchymal stem cells from human bone marrow, adipose tissue, and umbilical cord blood. Experimental Hematology. Volume 33, Issue 11, November 2005, Pages 1402–1416.
- 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
- Coleman CM., Curtin C., Barry F.P., . O’Flatharta C., Murphy JM. Mesenchymal stem cells and osteoarthritis: remedy or accomplice?. Hum Gene Ther, 21 (2010), pp. 1239–1250.
- Peng Xia, Xiaoju Wang, Qiang Lin, Xueping Li. (2015, Dec). Efficacy of mesenchymal stem cells injection for the management of knee osteoarthritis: a systematic review and meta-analysis. International Orthopaedics. Volume 39, Issue 12, 2363-2372
- Ishiguro N, Kojima T, Poole AR. Mechanism of cartilage destruction in osteoarthritis. Nagoya J Med Sci. 2002;65:73–84
- Blagojevic M., Jinks C., Jeffery A., Jordan K.P. (2010) Risk factors for onset of osteoarthritis of the knee in older adults: a systematic review and meta-analysis. Osteoarthritis Cartilage 18: 24–33
- Felson D.T., Lawrence R.C., Dieppe P.A., Hirsch R., Helmick C.G., Jordan J.M., et al. (2000) Osteoarthritis: new insights. Part 1: The Disease and its risk factors. Ann Intern Med133: 635–646
- Simon LS. Curr Rheumatol Rep. 1999 Oct;1(1):45-7
- Kapadia BH, (2014).The economic impact of periprosthetic infections following total knee arthroplasty at a specialized tertiary-care center. J Arthroplasty.
- Goldring MB. Chondrogenesis, chondrocyte differentiation, and articular cartilage metabolism in health and osteoarthritis. See comment in PubMed Commons belowTher Adv Musculoskelet Dis. 2012 Aug;4(4):269-85.
- Mueller MB , Tuan RS. Functional characterization of hypertrophy in chondrogenesis of human mesenchymal stem cells. Arthritis Rheum. 2008 May;58(5):1377-88.
- Harvey W.F., Hunter D.J. The role of analgesics and intra-articular injections in disease management. Rheum Dis Clin North Am, 34 (2008), pp. 777–788.
- Caplan A. What are MSCs therapeutic? New data: new insight. J Pathol. 2009;217:318–324. doi: 10.1002/path.2469.
- Van Buul GM., Villafuertes E., Bos PK., , Waarsing JH., Kops N., , Narcisi R., Weinans , J.A.N. Verhaar, , M.R. Bernsen, G.J.V.M. van Osch. Mesenchymal stem cells secrete factors that inhibit inflammatory processes in short-term osteoarthritic synovium and cartilage explant culture Osteoarthritis and Cartilage, 2012-10-01, Volúmen 20, Número 10.
- Caplan AI, Correa D. The MSC: an injury drugstore. Cell Stem Cell. 2011;9(1):11–15. doi: 10.1016/j.stem.2011.06.008.
- Caplan AI. Mesenchymal stem cells. J Orth Res. 1991;9(5):641–650. doi: 10.1002/jor.1100090504.
- Wu L, Leijten JC, Georgi N, et al. Trophic effects of mesenchymal stem cells increase chondrocyte proliferation and matrix formation. Tissue Eng. 2011;17(9-10):1425–1436. doi: 10.1089/ten.tea.2010.0517.
- de Windt T, Saris DB, Slaper-Cortenbach IC, et al. Direct cell–cell contact with chondrocytes is a key mechanism in multipotent mesenchymal stromal cell-mediated chondrogenesis. Tissue Eng Part A. 2015;21(19-20):2536–2547. doi: 10.1089/ten.tea.2014.0673.
- Puissant B., Barreau C., Bourin P., Clavel C. , Immunomodulatory effect of human adipose tissue-derived adult stem cells: comparison with bone marrow mesenchymal stem cells. Brithis Journal of Haematology. Volume 129, Issue 1. April 2005 . Pages 118–129
- Erickson, G.R.,Gimble, J.M.,Franklin, D.M., Rice, H.E., Awad, H. & Guilak, F. (2002) Chondrogenic potential of adipose tissue-derived stromal cells in vitro and in vivo. Biochemical and Biophysical Research Communications, 290, 763–
- Ming Liu, Martina M., Hutmacher D, Hoi Po Hui J., Hin Lee, Lim B. Identification of Common Pathways Mediating Differentiation of Bone Marrow- and Adipose Tissue-Derived Human Mesenchymal Stem Cells into Three Mesenchymal Lineages. Stem Cells. Volume 25, Issue 3. March 2007
- Marius Strioga, Sowmya Viswanathan, Adas Darinskas, Ondrej Slaby, and Jaroslav Michalek. Same or Not the Same? Comparison of Adipose Tissue-Derived Versus Bone Marrow-Derived Mesenchymal Stem and Stromal CellsStem Cells and Development. April 2012, 21(14): 2724- 2752.
- Gattazzo F., Urciuolo A., Bonaldo P. Extracellular matrix: A dynamic microenvironment for stem cell niche. Biochim Biophys Acta. 2014 Aug; 1840(8): 2506–2519.