|Year : 2014 | Volume
| Issue : 3 | Page : 134-142
Randomized blinded comparative analysis of autologous mononuclear medullary fraction and viscosupplementation in grade 0 and grade 1 radiographic scale of knee osteoarthritis
Sachin Upadhyay1, Hashmukh Shantilal Varma1, Sanyam Chaurasia1, Mayank Chansoria2
1 Department of Orthopaedics, Traumatology and Rehabilitation, Netaji Subhash Chandra Bose Medical College, Jabalpur, Madhya Pradesh, India
2 Department of Anaesthesiology, Netaji Subhash Chandra Bose Medical College, Jabalpur, Madhya Pradesh, India
|Date of Web Publication||11-Aug-2014|
622, "Poonam" Sneh Nagar, State Bank Colony, Jabalpur - 482 002, Madhya Pradesh
Source of Support: None, Conflict of Interest: None
Introduction: Bone marrow aspirate concentrate (BMAC) has been demonstrated to induce unfathomable healing activity in various forms of arthritis. On the other hand, viscosupplementation is widely used as one of the modalities to palliate the symptoms of knee osteoarthritis (OA). To the best of our knowledge, no research involving a comparison of the two interventions has been done. We evaluated and critically analyzed the comparative efficacy of an intra-articular injection of BMAC or sodium hyaluronate injection (Halonix; Cadila) in terms of pain relief or functional improvement using validated scoring instruments. Materials and Methods: Fifty patients of either sex, aged more than 18 years with grade 0 and grade 1 primary knee OA were randomized to receive intra-articular injection of either the mononuclear marrow concentrate or Halonix, and they were followed for 6 months. The patients treated received single injection either of mononuclear marrow concentrate or Halonix during the course of study. An independent, blinded evaluator assessed the patient with the Western Ontario and McMaster University Osteoarthritis Index (WOMAC), and the visual analog pain scale. Results: Both the cohorts treated with mononuclear marrow concentrate and with Halonix showed improvements from the baseline parameters at the end of 1-month follow-up. Group treated with BMAC showed significant difference (p<0.05) with respect to WOMAC, or visual analog scale results at the end of 6 months of follow-up. Men demonstrated significant improvement (p<0.05) in both the groups. Conclusion: Statistical significant differences were detected between patients treated with intra-articular injections of monocular marrow concentrate and those treated with the Halonix with respect to pain relief or function at 6 months of follow-up. Clinical Relevance: Direct delivery of bone marrow mononuclear concentrate to radiographic symptomatic grade 0 and grade 1 knee OA has the clinical potential to improve functional activity/ functional aspect of knee OA, providing a cost-effective approach for grade 0 and grade 1 knee OA.
Keywords: Bone marrow aspirate, knee osteoarthritis, viscosupplementation
|How to cite this article:|
Upadhyay S, Varma HS, Chaurasia S, Chansoria M. Randomized blinded comparative analysis of autologous mononuclear medullary fraction and viscosupplementation in grade 0 and grade 1 radiographic scale of knee osteoarthritis
. Indian J Pain 2014;28:134-42
|How to cite this URL:|
Upadhyay S, Varma HS, Chaurasia S, Chansoria M. Randomized blinded comparative analysis of autologous mononuclear medullary fraction and viscosupplementation in grade 0 and grade 1 radiographic scale of knee osteoarthritis
. Indian J Pain [serial online] 2014 [cited 2020 Apr 7];28:134-42. Available from: http://www.indianjpain.org/text.asp?2014/28/3/134/138437
| Introduction|| |
Symptomatic knee osteoarthritis (OA), presently the fourth major and the growing cause of disability worldwide,  has a formidable communal and public health impact.  An aging Asia with faster economic growth suggests greater prevalence of knee OA among other chronic conditions.  Several pharmacological/non-pharmacological therapies usually palliate the symptoms but these modalities only mask the problem. Knee arthroplasty is among the most frequently performed procedure in the developed world, and numbers are expected to rise as inhabitants age. Majority avoid surgery by using a variety of non-surgical treatments, including oral analgesics, non-steroidal anti-inflammatory drugs (NSAIDs), or intra-articular injections including corticosteroid, ,,,,, sodium hyaluronate, ,,, prolotherapy , and regenerative therapeutics. ,,,, One of the studies  showed that corticosteroids appear to be more effective than hyaluronic acid in both the short and long terms. While another  demonstrated no significant difference between these two drugs. The clinical utility of approaches based on regenerative therapeutics ,,,, show efficacy but unfortunately the astronomical cost and stringent requirements of these techniques has led to concern about their appropriate use especially in the context of developing countries. Furthermore, there is a scarcity of evidence in the peer-reviewed published literature on the comparative efficacy of regenerative therapeutic and viscosupplementation in improving clinical outcomes. The primary objective of the present pilot study is to test the hypothesis that there are significant differences between the intra-articular injections of stem cell-enriched mononuclear marrow aspirate concentrate or sodium hyaluronate injection in terms of relief in pain or functionality improvement as determined by validated scoring scales.
| Materials and Methods|| |
The study was designed as a single center prospective single blind randomized research. The present research was approved by institutional review board, and informed consent was obtained from each subject. A clinico-radiological evaluation of 80 subjects with knee pain was carried out from September 2010 to September 2011 who reported in the Department of Orthopedics. Weight-bearing anteroposterior radiographs were made for all patients prior to enrollment for the research. OA severity was scored using the Kellgren-Lawrence scale. 
Inclusion and exclusion criteria were defined. To be included, all participants aged 40-70 years of either sex had to (1) have been diagnosed as having primary knee OA and (2) have radiographic evidence (grade 0 and I; Lawrence and Kellgren radiological criteria) of severity.
Patients having severe OA (grade 2, 3 and 4); radiographic evidence of chondrocalcinosis; symptomatic insufficiency of knee ligaments; history of underlying inflammatory arthropathy or severe rheumatoid arthritis; Hyperuricemia (>440 μmol/l) and/or past history of gout; recent injury in the area affected by OA of the knee; intra-articular corticosteroid/LA injections within the preceding months; hypersensitivity to NSAIDs; abnormal liver or kidney function tests; history of peptic ulceration and upper gastrointestinal hemorrhage, congestive heart failure, hypertension, diabetes, cancer, hyperkalemia; major abnormal findings on complete blood count, history of coagulopathies, hematological or neurological disorders; and pregnant, breastfeeding or planning to become pregnant during the study were excluded from the research.
Among 80 subjects, 50 satisfied the inclusion criteria. The cohort included 12 men and 38 women. The mean age of the sample was 55.95 years (SD 8.446). Equally sized cohort were identified, each of the sample group having 25 subjects of either sex, were randomized into two clinical groups based on their serial number (odd or even) assigned at their reporting time to the outpatient department. All patients with even serial number were placed under group A (intra-articular injection of stem cell-enriched bone marrow aspirate concentrate; BMAC) and the patients with odd serial number were gathered under group B (intra-articular injection of Halonix). The patients treated received single injection either of mononuclear marrow concentrate or Halonix during the course of study. Either group could request to shift to the NSAIDs therapy at any time during the course of the study. No attempt in either group was made to discourage the use of NSAIDs during the study course and they could request and receive the NSAIDs. Data collection and critical evaluation using validated scoring instruments was performed by a senior faculty who was blinded to the treatment received by each patient. Demographic data, including age, sex and the use of NSAIDs during the study period were recorded. Patients were advised to avoid strenuous activity for 2 to 3 days with other precautions following the intra-articular injections. Follow-up was done at 1 month, 3 months, 6 months and on an as-needed basis thereafter. All of the follow-up was done at the outpatient department, Department of Orthopaedics, N.S.C.B Medical College.
The present study utilized the Heraeus Cryofuge 6000i, a large volume refrigerated floor standing centrifuge, which offers a top speed of 4,240 rpm (6010 × g); dimensions (hxwxd) 1,178 × 800 × 905 mm; temperature control range, −20 0 C to + 40 0 C [Figure 1].
Before the procedure, OA grades were assessed by X-rays (weight-bearing AP) as per Lawrence and Kellgren classification.
Aspiration of bone marrow [Figure 2]
|Figure 2: Aspiration of bone marrow: (a) infiltration of local anesthetic agent; (b) bone aspiration needle (Jamshidi needle); (c) aspiration of bone marrow; (d) aspirated marrow|
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With all aseptic precautions under local anesthesia (1% lidocaine without epinephrine) in prone position, through a stab incision over posterior iliac crest the bone marrow aspiration needle was advanced slowly through the incision. The beveled needle was securely placed within the marrow cavity/spongy bone  in between the outer and inner tables; the marrow aspirated into a Dispovan 20 ml plastic syringe (heparin rinsed). At a given depth, the needle is turned 45˚ during consecutive aspirations to re-orientate the bevel, thereby facilitating aspiration from bigger area. After the tip is swept around a full circumference, the needle was withdrawn proximally roughly 1 cm through the same insertion site, and successive aspirations were begun, repeating the same procedure.
Processing and density gradient centrifugation
We employed Ficoll-Paque technique of density gradient centrifugation for efficient isolation of the BMSCs (Bone marrow mesenchymal stem cells or stromal cells) enriched mononuclear cell fraction of bone marrow. This technique using the principle of density gradient centrifugation yielded a virtually red blood cell (RBC)-free bone marrow cell suspension as described by Wells et al. Ficoll-treated bone marrow seems to contain higher concentrations of uncommitted stem cells. Heparinized marrow was diluted 1:1 in phosphate-buffered saline (PBS) and carefully layered over the Ficoll-Paque gradient in approximately 1:2-2.5 ratio (Ficoll-Paque solution of 1.077 g/ml density; ~1:2.5  ) [Figure 3]. The bone marrow aspirate was then concentrated using the centrifugation system (Heraeus Cryofuge 6000i; [Figure 1]) for 30 min at 1,500 rpm at 22-24 0 C [Figure 1]. Density gradient principle settled the majority of the mononucleated cells and platelets on top of the Ficoll-Hypaque layer because they have a lower density; in contrast, RBCs and granulocytes at the bottom of the Ficoll-Hypaque layer [Figure 4]. The mononuclear cell interface layer was harvested carefully using long bore micro-pipette after discarding the supernatant. The resultant mononuclear cells were re-suspended twice in PBS (for 5 min at 1,500 rpm). The marrow concentrate was transferred by sterile technique back to the sterile field for intra-articular injection. Both aspirate and concentrate (1 mL were saved of each sample for in vitro analyses) were analyzed using flow cytometry (FACS) regarding the containing cells [Figure 5]. One drop of concentrate is taken in slide for staining with trypan blue dye (1:1) to assess the viability of mononuclear fraction. Sample is also sent for culture and sensitivity test. Rest is preserved for injection under IITV control. The diluted BMAC (2 ml) is injected slowly injected intra-articularly into the knee joint under image intensifier in group A patients under aseptic precautions.
|Figure 3: Layering of bone marrow in a test tube with lymphoprep: (a) lymphoprep (b) lymphoprep in conical tube; (c) bone marrow and lymphoprep in conical tubes; (d) layering of bone marrow over lymphoprep; (e) bone marrow layered over lymphoprep|
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|Figure 4: Showing density gradient sedimentation (moving downward: Clear layer of plasma; buffy-colored mononuclear concentrate; clear layer of Ficoll; RBCs and granulocytes)|
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|Figure 5: Flow cytometric analysis: Aspirate and concentrate showing significant concentration of medullary fraction (CD 34, CD45, Lymphs)|
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BMSCs are immature natural resident pluripotent cells of bone marrow with a wide spectrum of differentiation proliferative capacity. ,, Mesenchymal stromal cells (MSCs) have an inherent capability to differentiate into cells of three blastophyllums (ectoderm, mesoderm, and endoderm).  MSC-enriched mononuclear aspirate concentrate was delivered to the OA knee by direct intra-articular injection as technically it is the simplest mode of delivery. Since the progenitor cells cannot easily be identified within the medullary fraction without additional laboratory analysis, the complete mononuclear fraction has to be used. Following injection, the mononuclear concentrated dispersed throughout the joint cavity. In due course they would interact with the available receptors and surfaces.  The Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy provides minimum defining criteria for MSCs.  We don't have the facility for identifying the same in the institution; so in order to determine the concentration of mononuclear aspirate in the present research flow cytometric analysis [Figure 5] were done just to determine the subset of circulating cells expressing the CD34 antigen to assess the progenitor cell content in the whole marrow and after density gradient centrifugation i.e., in concentrate. Since it is very expensive, authors does not recommend routine clinical use of flow cytometry.
Injection of sodium hyaluronate
Under aseptic conditions, a prefilled 2-mL syringe [Figure 6], needle was placed supero-laterally into the suprapatellar pouch and slowly injected the content into the joint.
Data are presented as means ± SD. All calculations and statistics were performed with Statistical Package of Social Sciences (SPSS 20) software. A "p value" of less than 0.05 (p<0.05) was regarded as significant.
| Result|| |
Fifty patients with primary OA of the knees participated in this study. Of the participants, 12 (24%) were men and 38 (76%) were women [Table 1]. Twenty-five patients per treatment group had baseline measurements. A total of 11 patients (six in the sodium hyaluronate group and five in the BMAC group) showed premature discontinuation; data were available for 25 patients in the BMAC group and 25 in the Halonix group at baseline; for 24 and 23 patients, respectively, at 1 month; for 21 and 20 patients, respectively, at 3 months and for 20 and 19 patients, respectively at 6 months [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6].
|Table 1: Cohort characteristics (patients demographic; severity variables)|
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The present study showed significant preponderance of females (p<0.05) [Table 1]. A statistically significant difference was found between the two groups with respect to the radiographic evidence of the disease (p<0.05), with more patient with grade 1 arthritis in viscosupplementation group [Table 1]. In each treatment group, for the same grade of arthritis, women shows significant higher score than men on the baseline visual analog scale (p<0.05).
The scores on the visual analog scale improved from baseline for both groups [Table 2]. Hyaluronic acid was better but not significant (p>0.05) than BMAC group for pain relief at 1 month. However, at 6 months BMAC group showed a significant (p<0.05) benefit compared with the viscosupplementation group for the pain component. At 3 months of follow-up, group B patients show significant shifting to the NSAIDs therapy (p<0.05) [Table 1]. Group A patients showed significant reduction (p<0.05) in the NSAIDs usage during the study period in comparison to group B subjects. Mean stiffness score of WOMAC scale in group A gradually decreased from pre-injection value of 2.88 ± 0.68 to 0.85 ± 0.671 at 6th month follow-up and showed significant difference when compared with group B patients [Table 4]. Similarly, the mean Physical Function (Pf)/Activities of Daily Living showed statistical difference as compared to group B patients [Table 5]. The total WOMAC scores although better than baseline for both treatment groups, the patient treated with BMAC showed statistically significant improvement (p<0.05) at 6 months of follow-up [Table 6]. A total of 11 patients (six in the Halonix group and five in the BMAC group) patients did not complete the study (at 6 months). The reasons for premature discontinuation were a lack of efficacy (six patients in the Halonix group) and loss to follow-up (five patients in the BMAC group). There were no infections in the present series. Pain at the injection site was described by 60% (30) of the 50 patients, with no significant difference noted between the groups. This pain typically lasted no more than two days. Furthermore, there has been no hematoma formation or infection at the site of aspiration.
| Discussion|| |
OA of the knee presents a mounting burden on healthcare systems for which the therapeutic armamentarium remains inadequate and limited. We report the outcome of a prospective, blinded, randomized pilot research comparing the efficacy of intra-articular injections of the BMAC and Halonix (Cadila) in 50 patients with OA of the knee. To the best of our knowledge, no randomized clinical research comparing the efficacy of these two components has been published. Authors found that pain is critical in deciding patient's satisfaction , functional activity measures, and to some extent explicate the better results in favoring intra-articular injections of the stem cell enriched mononuclear marrow aspirate for these outcomes at 6 months of follow-up.
In the current study, females showed significantly less response to both treatments (p<0.05), which is in agreement with Leopold et al. study.  This could be attributed to the fact that female cartilage is subjected to more stress, as in Asian scenario, squatting, kneeling and sitting cross-legged activities are socioeconomic culture; more pronounced in females than in males that may account for greater functional disability. ,, Also, prior literature showed that women having greater sensitivity to heat, cold, and mechanical pressure. ,, Furthermore, the females showed reluctant attitude toward the precautions they have advised to follow up following intra-articular injection.
Several studies used MSCs enriched autologous bone marrow for chondrogenic repair. ,,,,, Density gradient centrifugation removes erythrocytes for the efficient isolation of the BMSC-enriched mononuclear cell fraction of the bone marrow. The rationale to deliver aspirate concentrate is to deliver higher concentration of mononuclear fraction containing progenitor's cells. In our setting, the centrifugation technique enriched population of bone marrow-derived cells by a factor of 7.82 (before 16.55 ± 0.5127/μL; after centrifugation, viable CD34 = 129.55 ± 10.01513 /μL (p<0.05)) [Figure 5]. An alternative approach to regenerative therapy is the injection of unfractionated whole bone marrow (WBM) into osteoarthritic joints.  Authors are against the use of WBM for two reasons. First and foremost is that this technique may be inefficient for the isolation of BMSCs because untreated bone marrow contains a large quantity of erythrocytes and their existence may impede with the initial adherence of BMSCs. Thus the removal of unnecessary high density blood component is critical by density gradient centrifugation or hemolysis so as to increase the number of colony-forming units (CFUs) in primary BMSC culture.  Second, blood in a joint is unphysiological and potentially destructive.  Single episode of hemarthrosis (traumatic or induced by giving intra-articular injection of blood) causes lasting damage to the articular cartilage. 
In the present group treated with sodium hyaluronate, relief of symptoms was appreciated as early as 4 weeks after treatment in agreement with Kotz and Kolarz  but it gradually weans off. This could be attributed to the fact that all injected hyaluronic acids are gone within days  and also due to inadequate dosing in the present study. Multiple injections have been thought to be essential for a longer-lasting (6 months to 1 year) effect on osteoarthritic pain. 
In subsequent follow-up period, more patients in the viscosupplementation group reported no significant benefits or improvement in outcomes. At 6-month follow-up, we found that patients treated with intra-articular injections of the BMAC had less pain, better functional activity and were more satisfied compared to a group of patients treated with viscosupplementation. The patients treated with sodium hyaluronate didn't show significant improvement from baseline at 6 months of follow-up period. The answer is probably for a combination of reasons : s0 ubjects with more severe grade of OA, inadequate dose of sodium hyaluronate, and most critical, the lack of regenerative capability of sodium hyaluronate. On the other hand, patients treated with enriched mononuclear fraction showed significant improvement in subsequent follow up. Currently, it is difficult to discern the underlying mechanism for the same but most probably it could be either direct due to their (BMAC) intrinsic ability for repair and regeneration or indirect through their immunomodulatory and paracrine effects. , The critical analysis of the current research showed that BMAC injection appears to have a slower onset of action than intra-articular viscosupplementation, but the effect seems to last longer.
The present study clearly demonstrates intra-articular stem cell-enriched BMAC injection to be an effective and well-tolerated alternative to viscosupplementation in the management of patients of radiographic evidence (grade 0 and 1) of knee OA with an added advantage of its biological nature and better patient compliance. The critical findings of present series will open up new avenues of research into a better understanding of the role of bone marrow mononuclear cell concentrate in OA. Authors feel that more scientifically rigorous evidence is needed before intra-articular injection with BMAC can be generally accepted as an effective treatment for OA of the knee. This will require multicenter, randomized clinical trial with bigger cohort.
In present pilot study, authors have found intra-articular injection of BMAC to be safe and effective for OA of the knee. We recommend additional studies on the use of BMAC for the treatment of knee OA to help corroborate our outcomes.
| Limitations|| |
While the findings of the current study could be applied in most instances, there were some important limitations.
- It is unclear whether only some or all progenitor cells in the concentrate differentiated into chondroblast and integrating normally once injected. It is also difficult to tell that how many stem cells are required to induce chondrogenic repair in vivo.
- Small sample size and short follow-up period further limit the generalization of findings of the present study.
- Inclusion of only grade 0 and grade 1 Kellgren and Lawrence radiological assessment of OA.
- No attempts have been made to measure the cartilage volume neither through imaging nor through histopathological analysis.
| Acknowledgement|| |
We want to acknowledge the doctors and senior colleagues for providing fruitful and critical comments on the draft of this paper.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]