The effectiveness of knee osteoarthritis treatment by arthroscopic microfracture technique in combination with autologous bone marrow stem cells transplantation

Abstract

OBJECTIVE:

We aimed to evaluate the efficacy of the treatment of knee osteoarthritis (OA) patients by using microfracture technique in combination with autologous bone marrow stem cell transplantation.

METHODS:

A clinical study was conducted between November 2011 and January 2015 and involved 46 patients (aged from 46 to 69) with primary knee OA grade II and III (according to Kellgren-Lawrence classification) at the Orthopedic Trauma Department, Vietnam-Germany Friendship Hospital. Patients were randomly assigned to receive knee arthroscopy and then bone-marrow stem cells from their pelvic bones via injection.

RESULTS:

The mean Visual Analogue Scale (VAS) score of present pain decreased from 5.68 before surgery to 1.7 24 months after surgery. The mean preoperative Knee Injury and Osteoarthritis Outcome Score (KOOS) was 36.34 ( $\pm$ 3.13), which increased to 74.62 ( $\pm$ 2.86) 24 months after surgery. On the MRI scans, the average Noyes score decreased from 12 ( $\pm$ 1.46) to 7 ( $\pm$ 1.50). Cartilage volume increased on average from 0.4512 ( $\pm$ 0.26) cm ${}^{3}$ to 0.5463 ( $\pm$ 0.29) cm ${}^{3}$ 12–24 months after surgery.

CONCLUSION:

Treatment of osteoarthritis by a combination of arthroscopic microfracture and transplantation of autologous bone-marrow stem cells was an invasive, safe and effective method which showed an improvement in the clinical symptoms (VAS score) and knee functions (KOOS points).

Keywords

Osteoarthritis bone marrow stem cell arthroscopy microfracture

1. Introduction

Knee osteoarthritis (OA) is a common chronic disease in elderly people, which affects the quality of life of patients [1]. Typical manifestation of knee OA is a loss of cartilage because of an imbalance between cartilage biosynthesis and degradation [2]. When cartilage is damaged or aged, cartilage is not able to restore itself due to a lack of circulation. Hence, knee OA treatment (in OA early stage) is the only symptomatic treatment [3]. When knee OA advances to the late stage, the internal treatment is no longer effective, and joint replacement is one of the most common treatment options. However, knee replacement is a big and expensive surgery, with a high complications ratio and not all joints of patients with knee OA can be replaced [4]. Therefore, an exploration of methods to preserve the joint or prolong preoperative time is of utmost importance to clinicians. Bone marrow stem cells (SC) transplantation is a new development, which is already applied worldwide and has proven its effectiveness. In adults, bone marrow is located in the medullary cavity of long bones and in bony cavities of flat bones [5].

Worldwide there have been many preclinical and clinical trial studies demonstrating the ability to proliferate, differentiate into cartilage cells from the SC, helping to restore cartilage tissue. Gobbi and Whyte [6] reported the first cases of using mesenchymal SC from autologous bone marrow SC to treat knee OA. By 2011, Wakitani et al. [7] reported 45 cases of primary late-stage knee OA that were treated by autologous bone-marrow stem cell transplantation, with an average follow-up of 75 months (the longest was 11 years). Clinical symptoms significantly decreased at the time of claim and no patients in the study group had to undergo joint replacement surgery and no complications were found. Another study by Centeno et al. [8] was conducted on 339 degenerative knee joints, where the largest follow-up period up to 6 years, with similar results, without complications.

The purpose of this study is to determine the component and the quantity of bone marrow SC transplanted into knee OA and to evaluate the treatment results.

2. Materials and methods

2.1 Sample

The research sample included 46 patients, aged 46 to 69, with primary knee OA diagnosed according to the American College of Rheumatology 1991 [9], grades II and III (according to Kellgren-Lawrence classification) [10]. The treatment procedures were carried out at the Orthopedic Trauma Department, Vietnam-Germany Friendship Hospital, from November 2011 to January 2015.

Patients were excluded from the study if they were diagnosed with secondary knee OA, had trauma, axial malalignment, inflammation of the knee, rheumatoid arthritis, and other systemic diseases.

All knee OA patients signed a written consent prior to the study.

2.2 Study sample characteristics

Table 1
Study sample characteristics

Characteristics	$n$	%
Age group (mean age 54.82)
40–49 years old	8	17.4
50–59 years old	29	63
$>$ 60 years old	9	19.6
Body mass index (BMI)
Underweight ( $<$ 18.5)	0	0
Normal weight (18.5–22.9)	9	19.6
Overweight (23–24.9)	21	45.7
Class I obesity (25–29.9)	15	32.6
Class II obesity ( $\geqslant$ 30)	1	2.2
Site
Right side	29	62.5
Left side	17	37.5
Both sides	0	0
Main symptoms and signs
Knee pain	46	100
Morning stiffness ( $<$ 30 minutes)	38	82.6
Knee crepitus	42	91.3
Pivot shift	11	23.9
Level of osteoarthritis in radiography based on
Kellgren-Lawrence
Grade II	9	19.6
Grade III	37	80.4
Pre-operative VAS score	(Mean $\pm$ SD)
During activity	5.68 $\pm$ 0.37
Resting	2.77 $\pm$ 0.49
Pre-operative knee functions based on KOOS	(Mean $\pm$ SD)
Knee pain	42 $\pm$ 2.88
Other knee symptoms	42 $\pm$ 2.76
Daily activities	41 $\pm$ 2.76
Playing sports	24 $\pm$ 5.54
Quality of life	31 $\pm$ 3.95
MRI scan results	(Mean $\pm$ SD)
Mean Noyes score ( $n=$ 46)	12 $\pm$ 1.46
Mean cartilage volume (cm ${}^{3}$ ) ( $n=$ 23)	0.45 $\pm$ 0.26

Data on the research sample is presented in Table 1. The mean age of participants was 54.82 years (range 46 to 69). The most common age group included patients of 50 to 59 years of age (63%). The female-to-male ratio was 2:1 (i.e. more females than males). Patients with overweight and obesity accounted for 80.5% of the sample. Among patients operated, 62.5% had SC transplanted into the right knee and 37.5% had them placed into the left knee. All patients in this study reported a “knee pain” symptom, 91.3% had “knee crepitus”, 82.6% had morning stiffness and 11 patients or 23.9% experienced the “pivot shift” symptoms. Radiographic findings revealed that 80.4% of patients had osteoarthritis grade III (80.4%) and only 19.6% had grade II. VAS score for measuring pain differed between activity and rest. The mean VAS score during exercise was 5.68 $\pm$ 0.37, while at rest the mean VAS score was 2.77 $\pm$ 0.49. Among activities that involved the knee joint, sports and recreation were most popular, with the mean KOOS score of 24 $\pm$ 5.54. Pre-operative quality of life was disturbed, with the KOOS score of only 31 $\pm$ 3.95. The scores for knee pain, other knee symptoms, and function in daily living were quite similar (42 $\pm$ 2.88, 42 $\pm$ 2.76 and 41 $\pm$ 2.76, respectively). The MRI scan results showed that the Noyes score was 12 $\pm$ 1.46. The mean cartilage volume (of 23 inspected knees) was 0.45 $\pm$ 0.26 cm ${}^{3}$ .

2.3 Study design

Clinical trial without a control group.

2.4 Interventions

2.4.1 Bone marrow aspiration and SC extraction

Bone marrow aspiration was performed in the operating room under sterile conditions. Patients underwent spinal anesthesia while lying on stomach. 120 ml of bone marrow fluid was aspirated from the posterior iliac crest of both sides. It took about 15 minutes. The same surgical team performed all the procedures.

The bone marrow was transferred to laboratory to extract SC. The monocytes of the bone marrow were extracted by using density gradient centrifugation using Ficoll solution (density: 1.076). Bone marrow SC mass was generated by cell suspension in 10 ml of physiological saline.

The quality of SC was evaluated by counting cellular components. The number of haematopoietic SC was determined. The number of CD34 $+$ cells was counted using FACSCalibur (Becton, Dickinson and Company, USA). According to the principle of flow cytometry, the number of mesenchymal SC was determined by culture of colony forming unit-fibroblast (CFU-F). The latter was performed according to the procedure of Hematology Department, 108 Military Central Hospital. Bacterial and fungal cultures were analyzed.

Arthroscopic surgery making subchondral lesion (Microfracture Technique) was performed simultaneously with spinal anesthesia. After the bone marrow aspiration, the patient was placed on his/her back on the operating table; the patient’s position was the same as during normal knee arthroscopy. There was endoscopic cleansing of joints, with a creation of new holes on subchondral bones, 3–4 mm, 2–4 mm deep (Fig. 1). Different primary surgeons (but from the same surgical team) performed the arthroscopic surgical procedures and bone marrow aspiration.

Figure 1.

Creating new lesion on subchondral bones while tourniquet application (A, B); after creating lesion on subchondral bones and removal of tourniquet (C).

SC were injected into the joints immediately after knee arthroscopic surgery, with effective spinal anesthesia. The SC mass (10 ml) was injected entirely into the knee through the skin. After the injection, the knee was compressed with bandages, immobilized with a brace and with stretched posture.

Figure 2.

CFU-F cluster bottle (A), Fibroblast cell in one 1 CFU-F cluster (B).

2.4.2 Treatment and rehabilitation after surgery

Patients were treated after surgery with antibiotics, analgesics and edema reduction. Patients performed exercises for knee function rehabilitation during hospitalization and after being discharged from the hospital according to the protocol.

2.5 Evaluation of the results

Accidents and complications occurring during and after treatment were assessed.

The results of the surgery and the bone marrow SC injection were evaluated after 4, 6, 12, 18 weeks and after 24 months with the following indicators:

•
Knee pain assessment based on VAS (Visual Analogue Scale) score [11].
•
Knee function assessment based on KOOS (Knee Injury and Osteoarthritis Outcome Score) [12].
•
Knee restoration was evaluated on 3.0 Tesla MRI, postoperative images were scanned at 12–24 months. Change in cartilage thickness (change of Noyes score) was detected andt change in cartilage volume (measured by OsiriX software) was detected [13, 14]. The same group of experienced radiologists provided the MRI scan results and MRI cartilage measurement before and after treatment.

2.6 Data analysis

The results of the study were analyzed and processed using medical statistical algorithms, using the Stata 12.0 software, which included a paired sample $t$ -test (a comparison of two mean values for a group). Along with Student’s $t$ -test (two mean values of two groups were compared) and ANOVA (three or more values were compared). There was a test in quadrature and Fisher test.

2.7 Research ethics

A scientific council was consulted before conducting the research.

The patients were aware of the treatment objectives and accepted the possible risks. The study’s purpose was explained to patients as well as the possible benefits, risks and contributions to the science. The patients voluntarily participated in the study.

3. Results

3.1 Characteristics of SC block before transplantation

All the SC blocks were negative with bacteria and fungus. The type of cells in the stem cell block are listed in Table 2.

Generated SC blocks had 663.3 $\pm$ 461.2 $\times$ 10 ${}^{6}$ nucleated cells; 468.26 $\pm$ 306.24 $\times$ 10 ${}^{7}$ platelets; 8.15 $\pm$ 5.5 $\times$ 10 ${}^{6}$ CD34 ( $+$ ) cells. The quantity of CFU-F cell cluster (mesenchymal SC) was 33.34 $\pm$ 39.98 $\times$ 10 ${}^{3}$ (see Fig. 2).

3.2 Treatment outcomes

•
No early or late complications appeared in all participated patients.
•
Improvement in knee pain.

Table 2
Type of cells in stem cell block

Type of cells Unit Mean value Min Max

Total nucleated cells $\times$ 10 ${}^{6}$ 663.3 $\pm$ 461.2 644 2250

Total platelets $\times$ 10 ${}^{7}$ 468.26 $\pm$ 306.24 30 1300

Total CD34 ( $+$ ) cells $\times$ 10 ${}^{6}$ 8.15 $\pm$ 5.5 1.1 22.78

Total CFU-F $\times$ 10 ${}^{3}$ 33.34 $\pm$ 39.98 2.84 226.98

Table 3
Noyes score and cartilage volume in pre- and post-operation at 12–24 months with stem cell injection on MRI scan results

Pre-operation $+$ stem cell injection Post-operation $+$ stem cell injection at 12–24 months $p$

Mean Noyes scores 12 $\pm$ 1.46 ( $n=$ 46) 7 $\pm$ 1.50 ( $n=$ 36) $<$ 0.001

Mean cartilage volume (cm ${}^{3}$ ) 0.45 $\pm$ 0.26 ( $n=$ 21) 0.55 $\pm$ 0.29 ( $n=$ 21) $<$ 0.001

Figure 3.
Improvement of knee functions according to KOOS.

After 4 weeks of surgery, the VAS score in the active state did not change significantly ( $p>$ 0.05). At 6, 12, 18 and 24 months after surgery, the VAS score reduced significantly ( $p<$ 0.05). VAS score decreased soon, at 1 month after surgery in resting state ( $p<$ 0.05) and still decreased significantly during the next follow-up time.

•
Improvement of knee functions (Fig. 3).

Figure 3 shows that knee functions were improved significantly at 6, 12, 18 and 24 months ( $p<$ 0.05). Among activities involving knee joint, playing sports had the lowest score (and, accordingly, the slowest improvements of knee functions), i.e. 24 $\pm$ 5.54 before the operation, and 49 $\pm$ 5.11 after 24 months. There was an improvement in dealing with knee pain, with a pre-operative score of 42 $\pm$ 2.87, and 83 $\pm$ 2.14 after 24 months. Change in quality of life was also remarkable with the pre-operative score of 31 $\pm$ 3.95 and 78 $\pm$ 4.01 after 24 months.

KOOS improved gradually, from 36.34 ( $\pm$ 3.13) points up to 74.62 ( $\pm$ 2.86) points 24 months after having the operation and injection of bone marrow SC.
•
Improvement of cartilage on MRI scan results 12–24 months after surgery (Table 3).

Noyes score before treatment among 46 patients was 12 $\pm$ 1.46, Noyes score of 36 follow-up patients 12–24 months after surgery and SC injection was 7 $\pm$ 1.50, the change was statistically significant ( $p<$ 0.001). Pre-operative cartilage volume of 21 patients was 0.45 $\pm$ 0.2629 cm ${}^{3}$ , it improved significantly 12–24 months after surgery and SC injection with a score of 0.55 $\pm$ 0.29 cm ${}^{3}$ ( $p<$ 0.001).

4. Discussion

Type of cells	Unit	Mean value	Min	Max
Total nucleated cells	$\times$ 10 ${}^{6}$	663.3	$\pm$ 461.2	644	2250
Total platelets	$\times$ 10 ${}^{7}$	468.26	$\pm$ 306.24	30	1300
Total CD34 ( $+$ ) cells	$\times$ 10 ${}^{6}$	8.15	$\pm$ 5.5	1.1	22.78
Total CFU-F	$\times$ 10 ${}^{3}$	33.34	$\pm$ 39.98	2.84	226.98

	Pre-operation $+$ stem cell injection	Post-operation $+$ stem cell injection at 12–24 months	$p$
Mean Noyes scores	12 $\pm$ 1.46 ( $n=$ 46)	7 $\pm$ 1.50 ( $n=$ 36)	$<$ 0.001
Mean cartilage volume (cm ${}^{3}$ )	0.45 $\pm$ 0.26 ( $n=$ 21)	0.55 $\pm$ 0.29 ( $n=$ 21)	$<$ 0.001

The treatment of osteoarthritis was arthroscopic microfracture and transplantation of bone marrow SC. The advantages of these two methods were combined in the rehabilitation and regeneration of cartilage. Studies of biological features of bone marrow SC showed that besides being pluripotent, these cells also moved and accumulated principally in the injured cartilage. SDF1/CXCR signaling (appears in newly injured cartilage area) holds a crucial role in providing the pathway for the movement of SC to this area [15]. These bone marrow SC could also excrete a large amount of growth factor, cytokines, chemokines, commonly called modulate shift, and have different functions such as anti-inflammatory, anti-tissue-destructive, anti-fibrosis, osteoclast and angiogenesis [16]. The complex interaction between these mediators (which were secreted by SC) was proved to play an important role in mediating the proliferation, regeneration of many damaged organs, though the mechanism needs to be clarified. The lack of oxygen in the knee joint (the preference of cartilage living in an anaerobic environment) also inhibits collagen type X; cartilage itself is also a factor, which inhibits the osteoclast, fibrosis. Thus, the formation of cartilage is motivated and the formation of bone and fibrous is suppressed [17]. Arthroscopic microfracture creates a new injury in injured cartilage, on the one side, it creates activated factors, modulates shift, proliferation and differentiation activities of SC. On the other side, it creates a scaffold for adhesion of SC, the scaffold is a fibrin network formed in the area under the new injured cartilage after the dissolve of thrombosis. Studies about SC transplantation for cartilage regeneration showed that the proliferation, differentiation and shift of SC depend on cytokines, growth factor and matrix (buffer) where SC was inserted. Buffer herein may be fibrin, hydrogen, extracellular matrix or scaffold. Cytokines and other growth factors were created from alpha particle after thrombolysis or from a new injury. Platelet is supposed to contain protein (a kind of growth factor) which motivates the activity of SC, speeds up of wound healing including PDGF (platelet derived growth factor), VEGF (vascular endothelial growth factor) and TGF (transforming growth factor) [18, 19]. In this research, SC were activated by platelets in the new injury and platelets in bone marrow SC, with a mean density of 468.26 $\pm$ 306.24 G/L (Table 2). Thus, arthroscopic microfracture creates a pathway for the healing of injured cartilage, in which the fibrin acts as a scaffold for SC adhesion and at this site, under the serve of activators of such as platelets, growth factors, cytokines (appears at the injured site), SC could proliferate and differentiate into cartilage. If only SC participated in cartilage healing, the new cartilage would have a low amount of cell but rich in fibrous, more specifically fibrocartilage, which is biologically unstable and starts degenerating and fracturing after 48 weeks like in the Shapiro’s experiment. When transplanting (injecting) a large volume of bone marrow SC into the knee joint after arthroscopic microfracture, Saw et al. saw that the rehabilitation of injured cartilage by hyaline cartilage, with long-term knee function improved [20].

After the surgery and transplantation of bone marrow SC into the knee joint, no patient suffered from hematocele, bleeding in the site of bone marrow fluid separation or infection. No patients showed systemic or local reactions after SC injection. Risks of transplant rejection were prevented. Thus, the latter was an advantage of transplantation of autologous bone marrow SC. These results were similar compared to other studies [15, 21, 22, 23]. After at least 12 months (100%) and at most 24 months (58.7%) after surgery, no patient had signs of pain or any complaint in the site of bone marrow fluid separation and in the site of incision for knee arthroscopy. After a long follow-up time, no patient suffered from infection, inflammation, leakage, fibrosis or lumps in the knee. Two few-millimeters long surgical scars did not affect the functionality and aesthetics of the knee. It could be concluded that arthroscopic microfracture and transplantation of autologous bone marrow SC in the treatment of osteoarthritis is safe and less invasive. In the resting state, knee pain was decreased after 1 month of surgery, VAS score reduced from 2.77 to 2.37 ( $p<$ 0.05). In the active state, after 1 month of surgery, VAS score reduced from 5.68 to 5.61 ( $p>$ 0.05). After 6 months of surgery, knee pain was remarkably reduced in both resting state and active state. After 6, 12, 18 and 24 months of surgery, VAS score reduced significantly ( $p<$ 0.05) (see Fig. 1). The authors used the KOOS scale to assess the knee functions. The higher the KOOS points, the higher the knee functions. Average KOOS points increased from time to time, with the highest improvement in 6 months after surgery ( $p<$ 0.001), and continued to increase after 12, 18 and 24 months ( $p<$ 0.01). For assessing functions and rehabilitation of cartilage in the knee MRI scan results, the authors used Noyes classification and measured the cartilage volume using OsiriX software. Pre-operative Noyes score was 12 $\pm$ 1.46, the average volume of the cartilage of the most serious injured knee area was 0.45 $\pm$ 0.2629 cm ${}^{3}$ ; after 12–24 months of surgery, average Noyes score was 7 $\pm$ 1.5, the average volume of cartilage was 0.55 $\pm$ 0.2961 cm ${}^{3}$ ( $p<$ 0.001) (see Table 3). Results showed that there was an improvement in the rehabilitation in the cartilage thickness at the injured site after treatment.

5. Conclusion

Treatment of osteoarthritis by a combination of arthroscopic microfracture and transplantation of autologous bone marrow SC was an invasive, safe and effective method which showed a reduction in clinical symptoms (VAS score) and improvement of knee functions (KOOS points). There was rehabilitation of cartilage thickness on MRI scan results of 12–24 months. However, future studies should include more patients as well as a control group to validate the role of bone marrow SC in the rehabilitation and regeneration of cartilage.

Footnotes

Conflict of interest

The authors declare that they have no conflict of interest.

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