The Regenerative Effect of IntraArticular Injection of Autologous Fat Micro-Graft in Treatment of Chronic Knee Osteoarthritis

The study started in 2010 to find the effect of autologous fat micrograft for osteoarthritis (OA); the result was published on normal animal’s model, in 10 patients, then in 80 patients with knee osteoarthritis, and the current study in 205 patients. The study was conducted at King Abdulaziz University Hospital (January 2012–October 2015); 80 adult patients were suffering from moderate to severe knee osteoarthritis. About 10–20 mL fat micrograft was prepared with liposuction and injected intra-articularly into the affected knee/s. The results revealed that pain improvement after the fat injection during rest and with activity with the visual analogue scale. The Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) indicated improvement, both in the three domains (pain, stiffness, and physical function) and in total. The use of intra-articular autologous fat micrograft is simple, safe, and effective for degenerative knee osteoarthritis.


Introduction
Fat grafting and its use in aesthetic and reconstructive surgeries are considered a state of art, but looking back at the history, it is just a revival in the techniques, which was described previously by others when Gustav Neuber on 1893 was the first to perform fat graft for orbital depression in human and Erich Lexer who is a skilled German orthopedic and plastic surgeon reported a variety of clinical uses of fat graft in management of knee ankylosis and fat graft wrapped around the tendon during tendon repair to prevent skin tendon adhesion and restore gliding [1,2].
The revolution in surgical specialty directed toward minimally invasive therapeutic modalities where endoscopic surgery replaced the open surgical operations; similarly, the recent discovery of the regenerative effect of fat micrograft due to presence of adipose-derived stem cells (ADSCs), cytokines, growth factors, pre adipocytes, and mature adipocytes led to a growing interest for the

Surgical therapy
Surgical therapy included minor and major surgical operations, but the recent use of the minimally invasive surgical procedure of intra-articular injection fat micrograft with the contained adipose-derived stem cells, which we studied on animal model followed by human joints, showed very satisfactory outcome; this modality of treatment is the main theme of this chapter which will be discussed in details.
The minor surgical procedures include arthroscopic joint lavage debridement, which demonstrated short-term symptom relief with more improvement when combined with marrow-stimulating microfracture and drilling procedures of articular surface; this improvement in joint functions would postpone the need for knee replacement [25].

The major surgical procedures
On the other hand, joint replacement as major surgical intervention is reserved for patients with failure of other modalities and in patients with joint end-stage disease, as joint implants have a finite life span (~10-15 years). After that a variety of complications might occur such as wear particle formation, which contribute to loosening which required revision surgery; therefore the use of artificial joints in young patients (e.g., <55 years) is associated with higher revision rates of this operation with its associated disadvantages as being a major procedure with complications, long hospitalization, absence from work, and high cost, which indicate the need to develop new treatment options. Therefore tissue engineering regeneration offers a long-term solution for repair of the affected tissue components of the joints such as the bone, ligament, and knee meniscus [26][27][28].

The stem cell line therapy
Osteoarthritis is an active disease process with an imbalance between the repair and destruction and degeneration of joint with poor intrinsic healing power and Tibia Pathology and Fracture 4 regeneration due to poor vascularization and absence of direct access to progenitor cells of bone marrow [29].
For many years, researchers have been seeking to understand the body's ability to repair and replace the damaged tissues; these researches led them to the discovery of the unique mesenchymal stem cell, which is partly responsible for maintenance and repairing of damaged connective tissues after injury. They can migrate toward injured tissues, where they display trophic effects of synthesis of proliferative, proangiogenic, and regenerative molecules. Mesenchymal stem cells undergo site-specific differentiation into a variety of connective tissues including cartilage, bone, fat, tendon, ligament, marrow stroma, and others, with its reparative and regenerative effects with anti-inflammatory and immunomodulatory actions via direct cell-cell interaction or secretion of bioactive factors, resulting in differentiation, stemness maintenance, self-renewal, prevention, and modification of progress of the disease [17,[30][31][32][33][34][35][36][37][38].
Mesenchymal stem cells can be isolated from several human sources other than the bone marrow and fetal tissues, including adipose tissue (ADSCs) with similar phenotypic characteristics but different propensities in proliferation and differentiation potentials, and provide an abundant and easily accessible source of stem cells [39][40][41][42][43][44][45][46].
With all these properties, MSCs are considered as an ideal source of cell therapy for different types of diseases including bone and joint diseases as reviewed by Liu et al. [3] as a review article about therapeutic application of MSCs for common bone and joint diseases, which include over 265 clinical trials of MSCs registered with clinical trial for knee osteoarthritis and other joint and bone diseases; they conclude that MSC is a promising prospect in clinical application for bone and joint diseases, without any reports of post application adverse immune side effects [5].

Animal and human researches on uses of MSCs in joints
With the growing interest of using MSCs as biological treatment for cartilage repair in arthritic joint diseases on different animal models where stem cells grown on different media scaffolds include synthetic or natural extracellular matrix, implantation of stem cells into the joints is either as invasive via arthroscopy with possible increased risk of infection or noninvasive intra-articular injection MSCs. These trials are summarized in Table 1 [6,[46][47][48][49][50][51][52][53][54][55][56][57][58][59].

Our animal trial
Our study started as an idea on 2010, when we plan to use autologous fat micrograft for treatment of osteoarthritis and we started by injecting fat micrograft into normal hind joints of sheep to determine the safety and effects of intra-articular injection of autologous fat micrograft, followed by observing the animal's activities in using their injected joints, and by examining any macroscopic or microscopic changes in the articular cartilage of the fat-injected joints compared to other similar    non-injected joint of the same animal; the study confirmed the safety, without any associated detrimental effects, on the joint tissues. Furthermore, it had positive microscopic findings as there was increase of the thickness of the articular hyaline cartilage layer with significant proliferation of chondrocytes including different mitosis stages (Figures 1 and 2, Table 2). Therefore, intra-articular injection of fat micrograft is an ideal minimally invasive choice for joint lubrication with high potential healing effects.

Our human trial
After the successful encouraging results of our previous animal study, which demonstrates the potential healing power and regenerative effect of autologous fat micrograft with its stem cells and all other study reports of clinical trials and publication by using mesenchymal stromal/stem cells for management of osteoarthritis, which offer a great hope for the treatment of osteoarthritic joints, we decided to evaluate the efficacy of fresh non-processed autologous fat micrograft with its ADSCs for management of osteoarthritic joints as prospective interventional clinical trial, which was conducted at King Abdulaziz University Hospital, Jeddah, Saudi Arabia, after obtaining the ethical approval from the local research and ethics committee, No. 822-12, according to latest vision of the Declaration of Helsinki. Over the period of 2012-2013, a preliminary clinical trial was conducted on 10 adult patients of both genders suffering from severe to moderate knee osteoarthritis with encouraging results as an effective and safe method for the treatment of knee osteoarthritis, then we expand the trial on 80 adult patients which confirm our previous finding, and then the clinical trial concluded with the final reporting to ethical committee on December 2016 [2,3,6,7,66].
But our work in utilizing this modality of treatment continued, and we are currently presenting the outcome in 205 adult patients (392 knee joints) who were managed and completed the required period of follow-up [7, 60-75, 77, 78].
The other studies were mainly revolving around the use of bone marrow or expanded adipose tissues and non-expanded autologous MSCs although some trials use allogenic MSCs. Most researchers focus on the use of intra-articular injections without the use of scaffolds or major surgeries since injections are more cost-effective, have little morbidity, and are a desirable way of treatment. The satisfactory outcome of our study over 10 years indicated that MSC treatment appears to be a good option for treatment of moderate to severe OA in the elderly; other studies reported similar results to ours in demonstrating promising prospect of cell therapy in many refractory diseases, including bone and joint diseases, in great improvement of pain, mobility, and other joint functions; these have high potential for clinical use in tissue engineering and regenerative and reparative medicine. Other studies found MSCs effective in cartilage healing; these trials are summarized in

Study guidelines and patient selection
• Patients: all patients were adult patients from both genders and were screened for eligibility to participate in the study; each patient underwent a complete medical history, a physical examination, and a full assessment of the joint.  • Informed written consent was obtained from each patient before treatment after explaining to him all about the study and this modality of treatment.
• Inclusion criteria: all cases of severe to moderate knee osteoarthritis, the changes to be confirmed by bilateral anterior-posterior standing and lateral supine radiographs involving one or both knees.
• Exclusion criteria: recent knee surgery, chronic opioid intake, bleeding disorders, malignant disease, congenital or traumatic deformity of the knee joint, and refusal of the patient to be included in the study.
• For the evaluation of patient, we used the visual analogue scale for pain assessment (on scale 0-10 cm line, 0 = no pain and 10 = worst imaginable pain) was explained to patients during the preoperative visit; visual analogue scale at rest and during activity was obtained. •

Anesthesia
The procedures were performed under controlled local anesthesia and sedation. Dexmedetomidine 0.7 mcg/kg/hour was administered intravenously as a sedative and pain reliever. Patients were monitored for heart rate, pulse oximetry, temperature, and noninvasive blood pressure.

Procedures
The surgical site of liposuction was carefully chosen based on the availability of fat and the patients' wishes. Liposuction was performed under complete aseptic technique and antibiotic coverage of cefuroxime 1.5 g IV one dose, 1 hour preoperative followed by 500 mg orally every 12 hours for 7 days. Fat harvesting was obtained using 10-hole, Olivaire blunt cannula (Pouret Medical, Clichy, France) with 1 mm tip attached to a 10 mL Luer-Lok syringe (Terumo, Auburn, WA, USA). Fifty milliliters of fat micrograft was collected and then left for thirty minutes to settle and separate into various layers; the upper and lower layers were removed, while the middle layer of fat was kept for intra-articular injection (Figure 1).
The surgical site was prepared and injected with 100-200 mL of tumescent solution. Solution was prepared by mixing 30-50 mL of 1% lidocaine and 0.5 mg (0.5 mL) of epinephrine in 449.5 mL of lactated ringers. The osteoarthritic knee joint was injected with autologous intra-articular fat micrograft 15-20 mL through the lateral approach according to the case in an amount that did not produce high pressure inside the joint and did not produce pain to the patients due to tension of the joint capsule.

Postoperative advice and care
• After operation, the patient received antibiotics at home for 1 week and on regular pain killer for 2 weeks and is to start walking immediately as early as possible and increase activity as tolerated.
• Stress the preoperative advice to reduce weight, improve diet regimen, and perform regular exercise especially aqua or hydrotherapy therapy to strengthen muscle with consequently more improvement of outcome of the procedure.
• All patients were followed up in the clinic on a regular basis every 1-2 weeks in the first month and then every 3 months to assess incidence of side effects, complications, pain evaluation, stiffness and knee function problems, and recurrence of pain.
• The patient was informed that the improvement will start during the first month and increase with time, and the maximum appreciated improvement at 6 months, provided he will follow the given instructions and improve the predisposing risk and comorbid factors.

Statistical analysis
IBM SPSS Statistics for Windows, Version 20 (IBM Corp., Armonk, NY USA), was used for data analysis. Data were presented as mean ± SD and minimummaximum or number and percentage (n, %) as appropriate. Wilcoxon test for nonparametric variables was used to compare preinjection to postinjection values. A probability of ≤0.05 was considered significant.

The current study outcome of 205 patients
In this current study, we used the same methodology and patient's selection that we applied in the preliminary trial and in the main study of 80 patients indicated in the requested ethical approval, but in this chapter, we are presenting our experience in the management of 205 patients. Table 4 showed the demographic data and the clinical characteristics of the patients. The median age of the patients was 61.59 years, and the body mass index was 35.10 kg/m 2 . The female patients were more than male (74.10% versus 25.90%) with a ratio of 2.88:1. Only five patients (2.90%) were smoking. The associated comorbidities were obesity (74.60%), hypertension (34.60%), diabetes mellitus (21.50%), hypothyroidism (6.80%), rheumatoid arthritis (4.90%), low back pain (4.90%), hepatitis (2.00%), and lower limb edema (1.50%).   The duration of OA ranged from 1 to 33 years. The right knee was affected in 6.30% of patients and left knee in 2.40%, while both knees were affected in 91.20% of the cases. 99.50% of patients used NSAID, while glucosamine was used by 8.80%, prednisone by 4.90%, methotrexate by 3.40%, and relaxon by 4.40%. The number of fat injection was single in 97.10%, twice in 2.4%, or triple in 0.50% of cases ( Table 5).
VAS values were significantly higher in preinjection versus postinjection both during rest (8.02 ± 1.81 versus 0.69 ± 0.64, p < 0.0001) and with activity (9.53 ± 0.88 versus 1.46 ± 0.80, p < 0.0001) which reflected a highly significant improvement in OA pain ( Table 6 and Figure 3). Table 7 presented the Western Ontario and McMaster Universities Osteoarthritis Index before and after intra-articular fat micrograft injection. The three domains of WOMAC index, pain, stiffness, and physical function, were significantly lower in the post intra-articular fat injection period than the preinjection values. The total score of WOMAC test and its percentage were significantly lower in the post intra-articular fat injection period than the preinjection values (77.65 ± 11.84 versus 5.69 ± 4.60, p < 0.0001; 80.89 ± 12.34 versus 5.93 ± 4.79, p < 0.0001) ( Table 7 and Figures 4-7).  Table 6. Visual analogue scale values at rest and with activity before and after intra-articular fat micrograft injection.        Wilcoxon test for nonparametric variables was used to compare pre-to postinjection values.

Complications
We did have complication like infection or graft rejection; it was well tolerated because it is autologous.

Conclusion
Over 10 years our clinical study of treatment of chronic osteoarthritis using intra-articular injection of autologous fat micrograft offers an effective and safe treatment as a nonantigenic, lubricating, regenerative, and reparative modality which helps to restore the damaged cartilages and in turn improve joint pain, mobility, and other functions of the osteoarthritic joints; it is minimally invasive, without scars, and with lower cost than other lines of therapy, improves the quality of life, and is mostly effective with single injection, but reinjection is needed in some patients according to disease severity and chronicity. We found a selection of patients and preoperative correction of risk factors, e.g., obesity muscle weakness led to better outcome of the procedure.