Scholarly article on topic 'Effect of intra-articular injection of mesenchymal stem cells in cartilage repair in experimental animals'

Effect of intra-articular injection of mesenchymal stem cells in cartilage repair in experimental animals Academic research paper on "Medical engineering"

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Intra articular injection / Mesenchymal stem cells (MSCs) / Cartilage repair / Osteoarthritis / Experimental animals

Abstract of research paper on Medical engineering, author of scientific article — Nadia Salah Kamel, Mona Mahmoud Arafa, Amr Nadim, Hnaa Amer, Irene Raouf Amin, et al.

Abstract Osteoarthritis (OA) is characterized by degeneration of the articular cartilage and, ultimately, joint destruction. Human umbilical cord blood (hUCB) may prove to be a new source of mesenchymal stem cells (MSCs) for cellular therapeutics used for cartilage repair. Aim of the work This study was carried out over a nine-month period of time, to study the effect of intra-articular injection of hUCB MSCs in cartilage repair by histopathological and ultra structural assessment. Materials and methods We conducted our study on 20 adult rats, which were subjected to the induction of cartilaginous defect in both knee joints. This was followed by injection of MSCs suspended in Hyaluronic acid solution in the right knee of each rat while the left knee served as a control. Histopathological and electron microscopic studies were performed. Results The present study revealed: In the injected knees; In 73% of the cases, the tissue was typical of fibrohyaline cartilage and appeared more cellular than fibrous. In 27% of the cases the repaired tissue appeared more fibrous than hyaline. In the control knees; the newly formed tissue was an undifferentiated connective tissue and the cells were covered with a thin layer of fibrous tissue. The electron microscopic pictures of the injected knees showed mitotic chondrocyte activity. The pictures indicated a repaired fibrohyaline cartilage. Conclusion We can conclude that the intra-articular injection of hUCB MSCs is an effective method for cartilage repair in rats. This makes it a very promising tool for the treatment of patients with OA.

Academic research paper on topic "Effect of intra-articular injection of mesenchymal stem cells in cartilage repair in experimental animals"

The Egyptian Rheumatologist (2014) xxx, xxx-xxx

Egyptian Society for Joint Diseases and Arthritis The Egyptian Rheumatologist

www.rheumatology.eg.net www.sciencedirect.com

ORIGINAL ARTICLE

Effect of intra-articular injection of mesenchymal stem cells in cartilage repair in experimental animals

Nadia Salah Kamel a, Mona Mahmoud Arafa a, Amr Nadim d, Hnaa Amer b, Irene Raouf Amin a'*, Naglaa Samir c, Amina Salem a

a Department of Physical Medicine, Rheumatology and Rehabilitation, Faculty of Medicine, Ain Shams University, Egypt b Department of Clinical Pathology, Faculty of Medicine, Ain Shams University, Egypt c Department of Pathology, Faculty of Medicine, Ain Shams University, Egypt d Department of Obstetrics and Gynecology, Faculty of Medicine, Ain Shams University, Egypt

Received 10 February 2014; accepted 8 March 2014

KEYWORDS

Intra articular injection; Mesenchymal stem cells (MSCs);

Cartilage repair; Osteoarthritis; Experimental animals

Abstract Osteoarthritis (OA) is characterized by degeneration of the articular cartilage and, ultimately, joint destruction. Human umbilical cord blood (hUCB) may prove to be a new source of mesenchymal stem cells (MSCs) for cellular therapeutics used for cartilage repair.

Aim of the work: This study was carried out over a nine-month period of time, to study the effect of intra-articular injection of hUCB MSCs in cartilage repair by histopathological and ultra structural assessment.

Materials and methods: We conducted our study on 20 adult rats, which were subjected to the induction of cartilaginous defect in both knee joints. This was followed by injection of MSCs suspended in Hyaluronic acid solution in the right knee of each rat while the left knee served as a control. Histopathological and electron microscopic studies were performed.

Results: The present study revealed: In the injected knees; In 73% of the cases, the tissue was typical of fibrohyaline cartilage and appeared more cellular than fibrous. In 27% of the cases the repaired tissue appeared more fibrous than hyaline. In the control knees; the newly formed tissue was an undifferentiated connective tissue and the cells were covered with a thin layer of fibrous tissue. The electron microscopic pictures of the injected knees showed mitotic chondrocyte activity. The pictures indicated a repaired fibrohyaline cartilage.

* Corresponding author. Tel.: +20 226223833.

E-mail address: irene_raouf@yahoo.com (I.R. Amin).

Peer review under responsibility of Egyptian Society for Joint Diseases

and Arthritis.

Elsevier I Production and hosting by Elsevier

1110-1164 © 2014 Production and hosting by Elsevier B.V. on behalf of Egyptian Society for Joint Diseases and Arthritis. http://dx.doi.org/10.1016/j.ejr.2014.03.001

Conclusion: We can conclude that the intra-articular injection of hUCB MSCs is an effective method for cartilage repair in rats. This makes it a very promising tool for the treatment of patients with OA.

© 2014 Production and hosting by Elsevier B.V. on behalf of Egyptian Society for Joint Diseases and

Arthritis.

1. Introduction

Osteoarthritis (OA) is a degenerative joint disease that is characterized by erosion of the articular cartilage, growth of bone at the margins i.e., osteophytes, subchondral sclerosis and a range of biochemical and morphologic alterations of the synovial membrane and joint capsule [1]. The primary tissue affected is the thin rim of hyaline articular cartilage interposed between the two articulating bones [2].

In early OA, the articular cartilage surface becomes irregular, and superficial clefts within the tissue become apparent. As the condition worsens, the clefts deepen, surface irregularities increase and the articular cartilage eventually ulcerates, exposing the underlying bone [3]. Chondrocytes in areas surrounding an injured zone are unable to migrate, proliferate, repopulate, regenerate or repair tissue with similar structure, function, and biomechanical properties of normal hyaline cartilage [4].

The burden of OA is exacerbated by the inadequacies of current therapies. Nonpharmacologic and pharmacologic treatments are used for early and moderately early cases of OA [5].

Mesenchymal stem cells (MSCs), which have the ability to differentiate into cells of the chondrogenic lineage, are very promising candidates to develop new cell-based articular cartilage repair strategies; this strategy entails the use of MSCs as trophic producers of bioactive factors to initiate endogenous regenerative activities in the OA joint [6]. Autologous bone marrow (BM) represents the main source of MSCs for both experimental and clinical studies; however as the number of MSCs and their differentiation capacity decline with age, their therapeutic potential might be diminished as well [7]. As several ethical and practical issues arise from the use of BM and fetal stem cells, umbilical cord blood (UCB) has turned out to be an excellent alternative source of MSCs for clinical-scale allogeneic transplantation [8].

It was postulated that hyaluronic acid might facilitate the migration and adherence of MSCs to the defect, which might explain the occurrence of partial healing at 6 weeks in animals that were treated with hyaluronic acid alone. The repaired tissue in animals treated with hyaluronic acid alone was of inferior quality and was shown to deteriorate further after 12 weeks, so the combination between MSCs and hyaluronic acid will result in synergistic effect [9].

Aim of the Work: This study was carried out over a nine-month period of time, to study the effect of intra-articular injection of human umbilical cord blood (hUCB) mesenchy-mal stem cells (MSCs) in cartilage repair by histopathological and ultra structural assessment.

2. Materials and methods

The study was carried out on twenty albino rats of Wistar strain (adult males) during a nine-month period of time. They

were brought from the medical research center, faculty of medicine, animal house of Ain Shams University. This study was approved by the local ethical committee.

The animals were maintained under conditions of controlled humidity, they were fed with commercial rat pellets and water. The animal house staff detected the age and weight of the animals and supervised their feeding. All animals were healthy and had no joint problems.

2.1. All animals were subjected to the following

Induction of cartilaginous defect in both knee joints by scratching the cartilage using a sterile needle [10,11]. In maximal flexion, longitudinal and diagonal grooves were made on the weight-bearing parts of femoral condyles without damaging the subchondral bone. The latter was checked by histology in 2 of the rats which were sacrificed; one of them after 1 week and the other after 4 weeks from the scratching which was done in the beginning of the experiment in order to ensure development of osteoarthritis (OA).

2.2. Preparation of MSCs from human umbilical cord blood (UCB)

2.2.1. Collection of UCB

Umbilical cord blood was obtained from the labor room of Obstetrics and Gynecology Department, Faculty of Medicine, Ain Shams University after written consent from the mothers.

4 UCB samples from full-term deliveries were collected from the unborn placenta using complete aseptic technique in sterile 15 ml Falcon tubes (Nunclon, Germany) containing 2 ml of acid citrate dextrose (ACD) anticoagulant (Lonza, Switzerland). The samples were stored at 22 ± 4 0C before processing [12]. Isolation and culture of MSCs were carried out in the medical research center, Faculty of Medicine, Ain Shams University as follows.

2.2.2. Isolation of mononuclear cells (MNCs)

Under complete asepsis each UCB sample was diluted 1:1 with phosphate-buffered saline (PBS) (lonza) and was carefully loaded onto Ficoll-Hypaque solution 2:1 ratio. After density gradient centrifugation at 2000 rpm for 30 min at room temperature, MNCs were removed from the interphase (the Buffy coat) and were washed three times with PBS; each time were centrifuged at 1500 rpm for 5 min, a clear cell pallet was formed in the bottom of the tube [13].

2.2.3. Culture of mesenchymal stem cells

The cells were cultured in complete culture medium; Dulbecco's modified Eagle's medium (DMEM) (Lonza, Switzerland) containing 12% fetal calf serum, 1% antibiotics -antimycotic; 100 units/ml of penicillin, 100 ig/ml of streptomycin and 250 ig/ml Amphotericin B. Cells were

placed in 25 ml Falkon flasks (Nunclon, Germany). The flasks were incubated at 37 0C in 5% CO2 (NuAire, USA).

On the fifth day of culture, non-adherent cells were discarded and adherent cells were examined microscopically for morphological evaluation of spindle shaped cells. The complete medium was changed and the flasks were reincubated. Flasks were examined every other day for MSCs (spindle shaped, fibroblast like cells).

After 2 weeks of culture, the adherent cells were almost confluent. Adherent cells were harvested using 0.25% trypsin for 5 min at 37 0C, 5 ml of medium was added to deactivate it. Cells were then counted with a haemocytometer. Cells were then collected in a 15 ml Falcon tube and centrifuged at 2000 rpm for 10min. The supernatant discarded and the sediment washed twice with PBS, and then centrifuged 1500 rpm for 5 min [14].

2.2.4. Preparing MSCs for injection

After isolating MSCs we suspended them in 2 ml of Hyaluronic acid solution (Hyalgan®, Sanofi Aventis, USA) at a density of 1.2-1.5 x 106 cells/ml.

2.3. Injection of animals

Injection of MSCs suspended in Hyaluronic acid solution was done in the right knee of each rat after sterilization with Betadine solution. The left knee served as a control [15].

2.4. Preparation of histological sections of the knee joint

Animals were sacrificed by intraperitoneal injection of a lethal dose of thiopental (50 mg/kg) after 6 weeks of injection and knee joints were removed. Histopathological study was done to examine chondrogenic regenerative changes of the animal articular cartilage after injection [16].

The total knee joints were removed and fixed for 4 days in 10% neutral formalin, decalcified by 5% formic acid in distilled water. The decalcifying solution was renewed every 48 h until softening of the tissues. The decalcified specimens were washed and dehydrated in ascending grades of alcohol, cleared in xylene and embedded in paraffin. From each paraffin block, longitudinal sections (5 im) were cut and stained with hematoxylin & eosin and Masson's trichrome (for detection of collagen fibers).

2.5. Experimental assessment

Our experimental assessment was done to detect the development of OA in the knees of the studied rats and this depended upon subjective observational parameters as limping and/or aggressive behavior denoting pain.

2.6. Histopathological assessment

Each stained section was examined with a light microscope to assess the following criteria for cartilage repair: (1) Tissue morphology (2) Surface architecture (3) Clustering of chon-drocytes (4) Thickening of repaired cartilage (5) Synovial cell proliferation and inflammation.

The control slides were examined to assess the following criteria for osteoarthritic changes: (1) Surface irregularities (2) Organization and hypertrophy of chondrocytes and (3) The degree of proliferation and inflammation of synovial cells.

2.7. Preparation for electron microscopic (EM) study

The right (injected) knee joint of one of the knees was opened anteriorly by cutting through the ligamentum patellae. The articular cartilage surface was immediately washed with saline. Washing continued while the knee was disarticulated. The soft tissue surrounding the femur was reflected proximally and the limb amputated at the middle of femoral shaft. Specimen thus consisted of the distal half of the femur.

2.8. EM preparation of the sample

First, the specimen was immersed in freshly prepared glutaral-dehyde, buffered with 0.1 M sodium cocodylate at pH 7.3.The specimen was then left for 4 h at a temperature of four degrees centigrade. The specimen was then washed for 2 h in the three changes of the same buffer. Then, it was post-fixed in 1% osmium tetroxide for two hours in the refrigerator. The specimen was then washed in three changes of the same buffer for half an hour in each change. After post-fixation, dehydration of the specimen was carried out through ascending grades of ethanol alcohol at four degrees centigrade as follows: 30 min in each of the following concentrations of ethanol alcohol, 50%, 70%, 80% and 95%. Then, we used three changes alcohol for 30 min. At room temperature, the specimen was treated with three changes of propylene oxide over a period of one and a half hours. The specimen was impregnated in a mixture of equal parts of epoxy resin mixture and propylene oxide for 8 h. Finally, specimen was embedded in pure resin and was left for 48 h in an oven at 60 0C for polymerization of the resin. Section cutting was done on an LKB ultramicrotome. Ultra thin sections were obtained and picked up on coated copper grids. The ultrathin sections were stained with urenyl acetate for 30 min, and then followed by lead citrate for 15 min. The stained ultrathin sections were examined under transmission electron microscope; Sumy electron optics (TEM-100 SEO) Ukraine, at 60 kV accelerating voltage.

Electron microscopic study was done to visualize mitotic pictures (newly formed chondrocytes and chondroblasts), also to assess the collagen fibers and the matrix arrangement.

2.9. Statistical analysis

Data collected were presented in the form of numbers and percentages.

3. Results

This study was carried out on twenty albino rats of Wistar strain (adult males). Their age ranged from 6 to 12 months and their weight ranged from 180 to 220 g. All the animals were subjected to induction of cartilaginous defect in both knee joints by scratching the cartilage using a sterile needle in order to develop OA. Experimental assessment, in the form of observation of limping and/or aggressive behavior denoting

pain, revealed development of OA in all the studied rats. The 2 rats which were sacrificed after 1 and 4 weeks from the scratching maneuver showed evidence of development of OA in the form of fibrillation of the surface, the chondrocytes were smaller and lying in parallel longitudinal rows, the synovial cells showed mild proliferation and mild infiltration by inflammatory cells.

After induction of OA by scratching, one of the animals died before being injected. After 6 weeks of scratching; injection of MSCs suspended in hyaluronic acid solution was done in the right knee of 17 rats and the left knee served as a control. After injection none of the animals was limping or aggressive.

The animals were killed after 6 weeks of injection and knee joints were removed. Two of the animal samples were used in the electron microscopic study and the rest of the 15 animal samples were used for the histopathological assessment.

3.1. Microscopic findings

In the control group (left knees) (Table 1): the area of the defect was partially filled with a fibrovascular granulation tissue, which united the wound edges (Fig. 1a). The newly formed tissue was composed of undifferentiated connective tissue cells covered with a thin layer of fibrous tissue. It was grossly distinguishable from the surrounding normal tissue. Cartilaginous sequestra surrounded by organized fibrous tissue were seen (Fig. 1b). All the control knees did not show any signs of cartilage repair.

In the injected group (right knees) (Table 2): no signs of OA such as osteophytes, cysts formation, cartilage erosion or synovial proliferation were observed in any of the knees; fibrocar-tilaginous mass was detected at the region of the defect by Masson's trichrome stain in 4 treated knees (27%). The tissue appeared more fibrous than hyaline. The surface became moderately fibrillated, the chondrocytes were smaller and lying in parallel longitudinal rows, the subjacent matrix was densely fibrous and the synovial cells showed mild proliferation and mild infiltration by inflammatory cells (Fig. 2). In the remaining 11 treated knees (73%): the tissue was typical of

Table 1 Histopathological findings of the control knees (Lt. side).

No Joint surface Chondrocytes Synovial cells

Irregularity Disorganization Hypertrophy Proliferation Inflammation

1 2 1 1 2 2

2 1 1 1 2 2

3 2 2 1 1 1

4 1 1 1 2 2

5 2 1 1 2 2

6 1 1 1 1 1

7 2 2 1 2 2

8 1 2 2 2

9 1 1 1 1 1

10 1 1 1 1 1

1 1 1 1 1 1

12 2 2 1 2 2

13 1 1 1 1 1

14 2 2 2 2 2

15 2 2 2 2 2

Normal = 0, Mild = 1, Moderate = 2, Severe = 3.

Figure 1 (a) Control knees: Granulation tissue at the site of articular injury (H&E x400).

fibro-hyaline cartilage and appeared more cellular than fibrous. These findings were confirmed by Masson's trichrome stain that demonstrated pink nuclei subjacent to the pale blue staining matrix and fine fibrous tissue network showing intense metachromasia that divided the chondrocytes into clusters (Figs. 3a-c).

3.2. Electron microscopic findings

As regards the EM study of the MSC-injected knees; it showed a repaired fibro-hyaline cartilage demonstrated by many chondrocytes and chondroblasts. The chondrocytes had centrally positioned nuclei surrounded by a rich rough endoplasmic reticulum (rER) and numerous mitochondria.

Figure 1 (b) Control knees: Organization and formation of cartilaginous sequestra surrounded by disorganized fibrous tissue (H&E x200).

The chondrocytes also showed chondrocyte mitotic activity. The matrix displayed fine collagen fibrils (Fig. 4a). In some areas thick bundles of collagen fibrils, which were sectioned both transversely and longitudinally, were seen. Individual fibrils displayed alternating transverse dark and light banding along their length (Fig. 4b). The pictures indicated a repaired fibro-hyaline cartilage.

4. Discussion

Osteoarthritis is a major cause of disability in the elderly; the prevalence of this disease is expected to increase dramatically over the next 20 years with aged population [17,18]. The burden of OA is exacerbated by the inadequacies of current therapies. Non-pharmacologic and pharmacologic treatments are used for early and moderately early cases of OA, but protection of articular cartilage has so far not been convincingly shown [5,19]. Surgical intervention is often indicated when

Figure 2 Injected Knee: Chondrofication of old granulation tissue (arrow) with fibrillated surface (star) (H&E x200).

the symptoms cannot be controlled and the disease progresses [20]. Whether arthroscopic lavage and/or debridement can provide symptomatic relief is unclear [21]. Consequently, patients with severe OA are currently excluded from these treatments [22].

Rats as animal models have a short life span, are available, cheap and well adapted to our climate, these characters made them ideal for this study. Animals used in this study were adult rats because the cartilage of adult animals is resistant to repair than in immature animals and therefore more similar to adult human subjects [23]. We excluded females because they usually get pregnant which may disturb the study by additional factors out of the scope of our study.

Scratching the cartilage has the advantages that it is easy to perform and it induces partial thickness cartilage defect that does not reach the subchondral bone, so there is no involvement of the vasculature. Consequently, progenitor cells in blood and marrow cannot enter the damaged region to influence or contribute to the reparative process to ensure that any chondral repair is entirely from the injected cells [10].

Table 2 Histopathological findings of the mesenchymal stem cells (MSCs) injected knees (Rt. Side).

No Tissue Surface architecture Chondrocyte Cartilage Synovial cells Dose of injected

morphology (irregularity) clustering thickening Proliferation Inflammation cells (million cell)

1 HF 2 2 2 1 1 1

2 F 1 2 1 2 2 1

3 HF 2 2 2 1 1 1

4 HF 2 2 2 1 1 1

5 HF 2 2 2 1 1 1

6 HF 2 2 2 1 1 1

7 F 1 2 1 1 1 5

8 HF 2 2 2 1 1 5

9 F 1 2 1 1 1 5

10 HF 1 2 1 1 1 5

11 HF 1 2 1 1 1 3

12 HF 1 2 1 1 1 3

13 F 1 2 1 2 3

14 HF 2 2 1 1 1 3

15 HF 2 2 2 1 1 3

Normal = 0, Mild = 1, Moderate = 2, Severe = 3. Fibro cartilage = F, Hyaline-Fibrocartilage = HF.

Figure 3 (a) Injected Knee: Fibrohyaline cartilage covering the articular surface (H&E x200).

Figure 4 (a) Chondrocytes surrounded by fine collagen fibrils (TEM X3000).

Figure 3 (b) Injected Knee: Mature chondrocyte with subjacent

fibrohyaline matrix (H&E x400). Figure 4 (b) Bundles of collagen fibrils (TEM x15,000).

Figure 3 (c) Injected Knee: Hypercellular hyaline-like cartilage in which the fibrous network divides the chondrocytes into clusters (Masson's trichrom x400).

Since most OA models use knee joints, an important consideration in the use of these models is the load-bearing pattern of the species being used [24]. So we did not prevent the animal from weight bearing after induction of the cartilage defect, to eliminate the rest factor that may share in the healing process and we started treatment after 6 weeks of scratching to ensure that the OA is well established. On the other hand, Yanai et al.

[25] established an animal model of large full-thickness articular cartilage defects in the knee of rabbits using a hinged fixator and considered joint distraction to be favorable for the regeneration.

Human umbilical cord blood (hUCB) MSCs have many advantages because of the immaturity of newborn cells compared with adult cells also could be more convenient for cell transplantation and be a more economical source of MSCs, than bone marrow MSC. Furthermore, hUCB provides no ethical problems for basic studies and clinical applications

[26]. The study of Erices et al. [27] provided strong evidence for the presence of circulating non-hematopoietic stem cells in hUCB. Rosada et al. [28] has reported that cord blood

multilineage cells are slower to establish in culture, have a lower precursor frequency and a lower level of bone antigen expression, and lack constitutive expression of neural antigens when compared with bone marrow, suggesting a more primitive population. The hUCB is easy to obtain and to store, and its use for hematopoietic stem cell transplantation does not require a close human leucocyte antigen match as reported by Gluckman [29].

Human UCB stem cells are xenograft cells. Rat UCB is difficult to obtain and the available quantity of rat UCB is very limited; however, hUCB is easy to obtain and is plentiful. In our experiment, we expected to evaluate only the possibility of UCB stem cells differentiated into chondrocytes in vivo. So, we used hUCB in the rat model. Umbilical cord blood may prove to be a new source of cells for cellular therapeutics for stromal, bone, and potentially, cartilage repair. Also it is a very promising source in Egypt because of the high birth rate. For all the above-mentioned reasons, cord blood MSCs were considered in our study as the most promising cell source for cartilage repair.

Due to the specific structure and functions of placenta, human extra-embryonic MSCs, including MSCs derived from the UCB represent stem cell types, which combine some properties of pluripotent embryonic stem cells with other properties of multipotent MSCs. They have immunoprivileged characteristics, posses a broader plasticity, and proliferate faster than adult MSCs [30].

The hUCB MSCs were delivered in this study by intra-articular injection, there are 2 different approaches to this. One is to implant cells directly with or without a suitable matrix or scaffold seeded with chondroprogenitor cells and signaling substances as in the study of Jackson and Simon [31]. The alternative is to differentiate stem cells in vitro and implant a mature construct. The ability of stem cells to differentiate and adhere to scaffolds such as matrices of hyaluronan derivatives and gelatin-based resorbable sponge matrices has been investigated and proven by Solchaga et al. [32], and Pon-ticello et al. [33] respectively.

Most studies presume that scaffolds are required for the regeneration of cartilage. It is ventured that loads and fluid movements would simply prevent cells from thriving where they are needed. However Barry [34] has shown that MSCs can survive and thrive without a scaffold and injected stem cells have been recovered in viable form in a goat knee with simulated arthritis.

In the present study hUCB MSCs were delivered by intra-articular injection in a suspension of hyaluronic acid (HA) solution without the use of a solid biomatrix 6 weeks after scratching the cartilage. Lee et al. [9] postulated that HA might facilitate the migration and adherence of MSCs to the defect.

Our choice of experimental animals to be rats, limited our ability to experimental assessment. It was limited to subjective observational parameters such as limping or aggressive behavior denoting pain, so we chose the histopathological assessment and ultrastructural study that were based upon histological objectives. Samples were stained with hematoxylin & eosin and Masson's trichrome.

In the present study, the repair of lesions was a variable mixture of fibrous tissue, fibrocartilage and hyaline-like cartilage. At 6 weeks at the control group; the area of the defect was filled with a fibrovascular granulation tissue, the newly formed tissue was an undifferentiated connective tissue covered with a thin

layer of fibrous tissue. Cartilaginous sequestra surrounded by organized fibrous tissue were seen. All control knees did not show any signs of repair (differentiated cartilage) denoted that there is no systemic parenteral trafficking through systemic circulation from the injected right knee to the other left knee. These findings coincide with those reported by Yanai et al. [25], Bos et al. [35], and Lee et al. [9]. However in this study the tissue appeared more fibrous than cartilaginous.

At the injected group; no signs of OA such as osteophytes, cysts formation, cartilage erosion or synovial proliferation, were observed in any of the knees these findings match with that of Lee et al. [9].

After 6 weeks of injection fibrocartilaginous mass was detected in 27% of cases. The tissue appeared more fibrous than cartilaginous and the surface layers and the cells were more typically fibrocartilaginous than hyaline. The surface became moderately fibrillated, the chondrocytes are smaller and lying in parallel longitudinal rows, the subjacent matrix was densely fibrous and the synovial cells showed mild proliferation and mild infiltration by inflammatory cells. In 73% of cases; the tissue appeared more cellular than fibrous. The thin fibrillated surface and the cells were typical of fibro-hyaline cartilage; these findings match with those of Yanai et al. [25], Yan and Yu [36], and Lee et al. [9].

Regarding the EM study; it showed a repaired fibrohyaline cartilage demonstrated by many chondrocytes and chondro-blasts. The pictures indicated a repaired fibrohyaline cartilage.

A recent study conducted by Nam and his colleagues [37], showed a superior role of injecting autologous bone marrow derived mesenchymal stem cells (BM-MSCs) to hUCB MSCs. This study demonstrated hyaline-like cartilage regeneration in the knees of goats receiving BM-MSCs after two full-thickness chondral 5 mm diameter defects were created. They suggested that supplementing intra-articular injections of BM-MSCs following bone marrow stimulation (BMS) knee surgery provides superior cartilage repair outcomes.

We can conclude from these results that the intra-articular injection of hUCB MSCs is an effective method for cartilage repair in rats. This makes it a very promising tool for the treatment of patients with OA.

Conflict of interest

Acknowledgement

The authors would like to express their gratitude to Awatef Mohamed (Veterinary Fellow of Biochemistry) and Larissa Inge (Electron Microscopy) without whom this work would not have been accomplished.

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