The Egyptian Rheumatologist (2014) xxx, xxx–xxx

Egyptian Society for Joint Diseases and Arthritis

The Egyptian Rheumatologist

www.rheumatology.eg.netwww.sciencedirect.com

ORIGINAL ARTICLE

Effect of intra-articular injection of mesenchymal

stem cells in cartilage repair in experimental

animals

* 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.

Production and hosting by Elsevier

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http://dx.doi.org/10.1016/j.ejr.2014.03.001

Please cite this article in press as: Kamel NS et al. Effect of intra-articular injection of mesenchymal stem cells in cartilage repair in experanimals, The Egyptian Rheumatologist (2014), http://dx.doi.org/10.1016/j.ejr.2014.03.001

Nadia Salah Kamela, 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, Egyptb Department of Clinical Pathology, Faculty of Medicine, Ain Shams University, Egyptc Department of Pathology, Faculty of Medicine, Ain Shams University, Egyptd 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.

imental

2 N.S. Kamel et al.

Please cite this article in press as: Kameanimals, The Egyptian Rheumatologist

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 char-acterized by erosion of the articular cartilage, growth of boneat the margins i.e., osteophytes, subchondral sclerosis and arange of biochemical and morphologic alterations of the syno-

vial membrane and joint capsule [1]. The primary tissueaffected is the thin rim of hyaline articular cartilage interposedbetween the two articulating bones [2].

In early OA, the articular cartilage surface becomes irregu-lar, and superficial clefts within the tissue become apparent. Asthe condition worsens, the clefts deepen, surface irregularities

increase and the articular cartilage eventually ulcerates, expos-ing the underlying bone [3]. Chondrocytes in areas surroundingan 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 ofOA [5].

Mesenchymal stem cells (MSCs), which have the ability to

differentiate into cells of the chondrogenic lineage, are verypromising candidates to develop new cell-based articular carti-lage repair strategies; this strategy entails the use of MSCs as

trophic producers of bioactive factors to initiate endogenousregenerative activities in the OA joint [6]. Autologous bonemarrow (BM) represents the main source of MSCs for bothexperimental and clinical studies; however as the number of

MSCs and their differentiation capacity decline with age, theirtherapeutic potential might be diminished as well [7]. As sev-eral ethical and practical issues arise from the use of BM

and fetal stem cells, umbilical cord blood (UCB) has turnedout to be an excellent alternative source of MSCs for clini-cal-scale allogeneic transplantation [8].

It was postulated that hyaluronic acid might facilitate themigration and adherence of MSCs to the defect, which mightexplain the occurrence of partial healing at 6 weeks in animals

that were treated with hyaluronic acid alone. The repairedtissue in animals treated with hyaluronic acid alone was ofinferior quality and was shown to deteriorate further after12 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 histopathologicaland 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

l NS et al. Effect of intra-articular(2014), http://dx.doi.org/10.1016/j

were brought from the medical research center, faculty ofmedicine, 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 weightof the animals and supervised their feeding. All animals werehealthy and had no joint problems.

2.1. All animals were subjected to the following

Induction of cartilaginous defect in both knee joints byscratching the cartilage using a sterile needle [10,11]. In

maximal flexion, longitudinal and diagonal grooves were madeon the weight-bearing parts of femoral condyles withoutdamaging the subchondral bone. The latter was checked by

histology in 2 of the rats which were sacrificed; one of themafter 1 week and the other after 4 weeks from the scratchingwhich 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 techniquein sterile 15 ml Falcon tubes (Nunclon, Germany) containing2 ml of acid citrate dextrose (ACD) anticoagulant (Lonza,

Switzerland). The samples were stored at 22 ± 4 �C beforeprocessing [12]. Isolation and culture of MSCs were carriedout in the medical research center, Faculty of Medicine, AinShams 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 carefullyloaded onto Ficoll–Hypaque solution 2:1 ratio. After densitygradient centrifugation at 2000 rpm for 30 min at roomtemperature, MNCs were removed from the interphase (the

Buffy coat) and were washed three times with PBS; each timewere centrifuged at 1500 rpm for 5 min, a clear cell pallet wasformed 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, Swit-

zerland) containing 12% fetal calf serum, 1% antibiotics –antimycotic; 100 units/ml of penicillin, 100 lg/ml ofstreptomycin and 250 lg/ml Amphotericin B. Cells were

injection of mesenchymal stem cells in cartilage repair in experimental.ejr.2014.03.001

Effect of intra articular injection of mesenchymal stem cells in cartilage repair in experimental animals 3

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

On the fifth day of culture, non-adherent cells were

discarded and adherent cells were examined microscopicallyfor morphological evaluation of spindle shaped cells. The com-plete medium was changed and the flasks were reincubated.

Flasks were examined every other day for MSCs (spindleshaped, fibroblast like cells).

After 2 weeks of culture, the adherent cells were almost

confluent. Adherent cells were harvested using 0.25% trypsinfor 5 min at 37 �C, 5 ml of medium was added to deactivateit. Cells were then counted with a haemocytometer. Cells werethen collected in a 15 ml Falcon tube and centrifuged at

2000 rpm for 10 min. The supernatant discarded and thesediment washed twice with PBS, and then centrifuged1500 rpm for 5 min [14].

2.2.4. Preparing MSCs for injection

After isolating MSCs we suspended them in 2 ml ofHyaluronic acid solution (Hyalgan�, Sanofi Aventis, USA)

at a density of 1.2–1.5 · 106 cells/ml.

2.3. Injection of animals

Injection of MSCs suspended in Hyaluronic acid solution wasdone in the right knee of each rat after sterilization withBetadine 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 andknee joints were removed. Histopathological study was doneto examine chondrogenic regenerative changes of the animalarticular cartilage after injection [16].

The total knee joints were removed and fixed for 4 days in10% neutral formalin, decalcified by 5% formic acid indistilled water. The decalcifying solution was renewed every

48 h until softening of the tissues. The decalcified specimenswere washed and dehydrated in ascending grades of alcohol,cleared in xylene and embedded in paraffin. From each

paraffin block, longitudinal sections (5 lm) were cut andstained with hematoxylin & eosin and Masson’s trichrome(for detection of collagen fibers).

2.5. Experimental assessment

Our experimental assessment was done to detect the develop-ment of OA in the knees of the studied rats and this depended

upon subjective observational parameters as limping and/oraggressive behavior denoting pain.

2.6. Histopathological assessment

Each stained section was examined with a light microscope toassess the following criteria for cartilage repair: (1) Tissue

morphology (2) Surface architecture (3) Clustering of chon-drocytes (4) Thickening of repaired cartilage (5) Synovial cellproliferation and inflammation.

Please cite this article in press as: Kamel NS et al. Effect of intra-articularanimals, The Egyptian Rheumatologist (2014), http://dx.doi.org/10.1016/j

The control slides were examined to assess the followingcriteria 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 openedanteriorly by cutting through the ligamentum patellae. Thearticular cartilage surface was immediately washed with saline.

Washing continued while the knee was disarticulated. The softtissue surrounding the femur was reflected proximally and thelimb 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.Thespecimen was then left for 4 h at a temperature of four degreescentigrade. 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 speci-men was then washed in three changes of the same buffer for

half an hour in each change. After post-fixation, dehydrationof the specimen was carried out through ascending grades ofethanol alcohol at four degrees centigrade as follows: 30 minin each of the following concentrations of ethanol alcohol,

50%, 70%, 80% and 95%. Then, we used three changes alco-hol for 30 min. At room temperature, the specimen was treatedwith three changes of propylene oxide over a period of one and

a half hours. The specimen was impregnated in a mixture ofequal parts of epoxy resin mixture and propylene oxide for8 h. Finally, specimen was embedded in pure resin and was left

for 48 h in an oven at 60 �C for polymerization of the resin.Section cutting was done on an LKB ultramicrotome. Ultrathin sections were obtained and picked up on coated copper

grids. The ultrathin sections were stained with urenyl acetatefor 30 min, and then followed by lead citrate for 15 min. Thestained ultrathin sections were examined under transmissionelectron 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 andpercentages.

3. Results

This study was carried out on twenty albino rats of Wistarstrain (adult males). Their age ranged from 6 to 12 months

and their weight ranged from 180 to 220 g. All the animalswere subjected to induction of cartilaginous defect in bothknee joints by scratching the cartilage using a sterile needlein order to develop OA. Experimental assessment, in the form

of observation of limping and/or aggressive behavior denoting

injection of mesenchymal stem cells in cartilage repair in experimental.ejr.2014.03.001

Figure 1 (a) Control knees: Granulation tissue at the site of

articular injury (H&E ·400).

4 N.S. Kamel et al.

pain, revealed development of OA in all the studied rats. The 2rats which were sacrificed after 1 and 4 weeks from the scratch-ing maneuver showed evidence of development of OA in the

form of fibrillation of the surface, the chondrocytes weresmaller and lying in parallel longitudinal rows, the synovialcells 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; injec-

tion of MSCs suspended in hyaluronic acid solution was donein 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 inthe electron microscopic study and the rest of the 15 animalsamples 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 granulationtissue, which united the wound edges (Fig. 1a). The newlyformed tissue was composed of undifferentiated connective

tissue cells covered with a thin layer of fibrous tissue. It wasgrossly distinguishable from the surrounding normal tissue.Cartilaginous sequestra surrounded by organized fibrous tissuewere 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 syno-

vial proliferation were observed in any of the knees; fibrocar-tilaginous mass was detected at the region of the defect byMasson’s trichrome stain in 4 treated knees (27%). The tissue

appeared more fibrous than hyaline. The surface became mod-erately fibrillated, the chondrocytes were smaller and lying inparallel longitudinal rows, the subjacent matrix was densely

fibrous and the synovial cells showed mild proliferation andmild infiltration by inflammatory cells (Fig. 2). In theremaining 11 treated knees (73%): the tissue was typical of

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

No Joint surface Chondrocytes

Irregularity Disorganization

1 2 1

2 1 1

3 2 2

4 1 1

5 2 1

6 1 1

7 2 2

8 1 2

9 1 1

10 1 1

1 1 1

12 2 2

13 1 1

14 2 2

15 2 2

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

Please cite this article in press as: Kamel NS et al. Effect of intra-articularanimals, The Egyptian Rheumatologist (2014), http://dx.doi.org/10.1016/j

fibro-hyaline cartilage and appeared more cellular thanfibrous. These findings were confirmed by Masson’s trichromestain that demonstrated pink nuclei subjacent to the pale blue

staining matrix and fine fibrous tissue network showing intensemetachromasia 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 manychondrocytes and chondroblasts. The chondrocytes hadcentrally positioned nuclei surrounded by a rich roughendoplasmic reticulum (rER) and numerous mitochondria.

Synovial cells

Hypertrophy Proliferation Inflammation

1 2 2

1 2 2

1 1 1

1 2 2

1 2 2

1 1 1

1 2 2

2 2 2

1 1 1

1 1 1

1 1 1

1 2 2

1 1 1

2 2 2

2 2 2

injection of mesenchymal stem cells in cartilage repair in experimental.ejr.2014.03.001

Figure 1 (b) Control knees: Organization and formation of

cartilaginous sequestra surrounded by disorganized fibrous tissue

(H&E ·200).

Figure 2 Injected Knee: Chondrofication of old granulation

tissue (arrow) with fibrillated surface (star) (H&E ·200).

Effect of intra articular injection of mesenchymal stem cells in cartilage repair in experimental animals 5

The chondrocytes also showed chondrocyte mitotic activity.The matrix displayed fine collagen fibrils (Fig. 4a). In someareas thick bundles of collagen fibrils, which were sectionedboth transversely and longitudinally, were seen. Individual

fibrils displayed alternating transverse dark and light bandingalong their length (Fig. 4b). The pictures indicated a repairedfibro-hyaline cartilage.

4. Discussion

Osteoarthritis is a major cause of disability in the elderly; the

prevalence of this disease is expected to increase dramaticallyover the next 20 years with aged population [17,18]. Theburden of OA is exacerbated by the inadequacies of current

therapies. Non-pharmacologic and pharmacologic treatmentsare used for early and moderately early cases of OA, but pro-tection of articular cartilage has so far not been convincingly

shown [5,19]. Surgical intervention is often indicated when

Table 2 Histopathological findings of the mesenchymal stem cells (

No Tissue

morphology

Surface architecture

(irregularity)

Chondrocyte

clustering

C

th

1 HF 2 2 2

2 F 1 2 1

3 HF 2 2 2

4 HF 2 2 2

5 HF 2 2 2

6 HF 2 2 2

7 F 1 2 1

8 HF 2 2 2

9 F 1 2 1

10 HF 1 2 1

11 HF 1 2 1

12 HF 1 2 1

13 F 1 2 1

14 HF 2 2 1

15 HF 2 2 2

Normal = 0, Mild = 1, Moderate = 2, Severe = 3. Fibro cartilage = F,

Please cite this article in press as: Kamel NS et al. Effect of intra-articularanimals, The Egyptian Rheumatologist (2014), http://dx.doi.org/10.1016/j

the symptoms cannot be controlled and the disease progresses

[20]. Whether arthroscopic lavage and/or debridement canprovide symptomatic relief is unclear [21]. Consequently,patients with severe OA are currently excluded from thesetreatments [22].

Rats as animal models have a short life span, are available,cheap and well adapted to our climate, these characters madethem ideal for this study. Animals used in this study were adult

rats because the cartilage of adult animals is resistant to repairthan in immature animals and therefore more similar to adulthuman subjects [23]. We excluded females because they usually

get pregnant which may disturb the study by additional factorsout 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 thatdoes not reach the subchondral bone, so there is no involve-ment of the vasculature. Consequently, progenitor cells inblood and marrow cannot enter the damaged region to influ-

ence or contribute to the reparative process to ensure thatany chondral repair is entirely from the injected cells [10].

MSCs) injected knees (Rt. Side).

artilage

ickening

Synovial cells Dose of injected

cells (million cell)Proliferation Inflammation

1 1 1

2 2 1

1 1 1

1 1 1

1 1 1

1 1 1

1 1 5

1 1 5

1 1 5

1 1 5

1 1 3

1 1 3

2 2 3

1 1 3

1 1 3

Hyaline-Fibrocartilage = HF.

injection of mesenchymal stem cells in cartilage repair in experimental.ejr.2014.03.001

Figure 3 (c) Injected Knee: Hypercellular hyaline-like cartilage

in which the fibrous network divides the chondrocytes into clusters

(Masson’s trichrom ·400).

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

fibrohyaline matrix (H&E ·400).

Figure 4 (a) Chondrocytes surrounded by fine collagen fibrils

(TEM ·3000).

Figure 3 (a) Injected Knee: Fibrohyaline cartilage covering the

articular surface (H&E ·200).

Figure 4 (b) Bundles of collagen fibrils (TEM ·15,000).

6 N.S. Kamel et al.

Please cite this article in press as: Kamel NS et al. Effect of intra-articularanimals, The Egyptian Rheumatologist (2014), http://dx.doi.org/10.1016/j

Since most OA models use knee joints, an important con-sideration in the use of these models is the load-bearing pattern

of the species being used [24]. So we did not prevent the animalfrom weight bearing after induction of the cartilage defect, toeliminate the rest factor that may share in the healing process

and we started treatment after 6 weeks of scratching to ensurethat 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 hingedfixator and considered joint distraction to be favorable for theregeneration.

Human umbilical cord blood (hUCB) MSCs have manyadvantages because of the immaturity of newborn cells com-pared with adult cells also could be more convenient for celltransplantation and be a more economical source of MSCs,

than bone marrow MSC. Furthermore, hUCB provides noethical 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 cellsin hUCB. Rosada et al. [28] has reported that cord blood

injection of mesenchymal stem cells in cartilage repair in experimental.ejr.2014.03.001

Effect of intra articular injection of mesenchymal stem cells in cartilage repair in experimental animals 7

multilineage cells are slower to establish in culture, have alower precursor frequency and a lower level of bone antigenexpression, and lack constitutive expression of neural antigens

when compared with bone marrow, suggesting a more primi-tive 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 reportedby Gluckman [29].

Human UCB stem cells are xenograft cells. Rat UCB is dif-

ficult to obtain and the available quantity of rat UCB is verylimited; however, hUCB is easy to obtain and is plentiful. Inour experiment, we expected to evaluate only the possibilityof UCB stem cells differentiated into chondrocytes in vivo.

So, we used hUCB in the rat model. Umbilical cord bloodmay prove to be a new source of cells for cellular therapeuticsfor 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 wereconsidered 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 someproperties of pluripotent embryonic stem cells with other prop-erties of multipotent MSCs. They have immunoprivilegedcharacteristics, 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 suitablematrix or scaffold seeded with chondroprogenitor cells and sig-naling substances as in the study of Jackson and Simon [31].

The alternative is to differentiate stem cells in vitro andimplant a mature construct. The ability of stem cells to differ-entiate and adhere to scaffolds such as matrices of hyaluronan

derivatives and gelatin-based resorbable sponge matrices hasbeen 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 fluidmovements would simply prevent cells from thriving wherethey are needed. However Barry [34] has shown that MSCs

can survive and thrive without a scaffold and injected stemcells have been recovered in viable form in a goat knee withsimulated 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 afterscratching 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 behav-ior denoting pain, so we chose the histopathological assess-ment 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 mix-

ture of fibrous tissue, fibrocartilage and hyaline-like cartilage.At 6 weeks at the control group; the area of the defect was filledwith a fibrovascular granulation tissue, the newly formed tissuewas an undifferentiated connective tissue covered with a thin

Please cite this article in press as: Kamel NS et al. Effect of intra-articularanimals, The Egyptian Rheumatologist (2014), http://dx.doi.org/10.1016/j

layer of fibrous tissue. Cartilaginous sequestra surrounded byorganized fibrous tissue were seen. All control knees did notshow any signs of repair (differentiated cartilage) denoted that

there is no systemic parenteral trafficking through systemic cir-culation 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 studythe 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 withthat of Lee et al. [9].

After 6 weeks of injection fibrocartilaginous mass was

detected in 27% of cases. The tissue appeared more fibrousthan cartilaginous and the surface layers and the cells weremore typically fibrocartilaginous than hyaline. The surface

became moderately fibrillated, the chondrocytes are smallerand lying in parallel longitudinal rows, the subjacent matrixwas densely fibrous and the synovial cells showed mild prolif-

eration and mild infiltration by inflammatory cells. In 73% ofcases; the tissue appeared more cellular than fibrous. The thinfibrillated 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 fibrohyalinecartilage 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 inthe knees of goats receiving BM-MSCs after two full-thickness

chondral 5 mm diameter defects were created. They suggestedthat supplementing intra-articular injections of BM-MSCs fol-lowing 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 cartilagerepair in rats. This makes it a very promising tool for the treat-

ment of patients with OA.

Conflict of interest

None.

Acknowledgement

The authors would like to express their gratitude to Awatef

Mohamed (Veterinary Fellow of Biochemistry) and LarissaInge (Electron Microscopy) without whom this work wouldnot have been accomplished.

References

[1] Herndon JH, Davidson SM, Apazidis A. Recent socioeconomic

trends in orthopedic practice. J Bone Joint Surg

2001;83A:1097–105.

[2] Felson DT. Osteoarthritis of the knee. N Engl J Med

2006;354:841–8.

[3] Bollet AJ. The biology of degenerative joint disease. Arthritis

Rheum 1971;14(1):144.

injection of mesenchymal stem cells in cartilage repair in experimental.ejr.2014.03.001

8 N.S. Kamel et al.

[4] Martin JA, Buckwalter JA. Aging articular cartilage chondrocyte

senescence and osteoarthritis. Biogerontology 2002;3:257–64.

[5] Gerwin N, Hops C, Lucke A. Intraarticular drug delivery in

osteoarthritis. Adv Drug Deliv Rev 2006;58:226–42.

[6] Kolf CM, Cho E, Tuan RS. Mesenchymal stromal cells. Biology

of adult mesenchymal stem cells: regulation of niche, self-renewal

and differentiation. Arthritis Res Ther 2007;9:204.

[7] Mueller SM, Glowacki J. Age related decline in the osteogenic

potential of human bone marrow cells cultured in three-dimen-

sional collagen sponges. J Cell Biochem 2001;82:583–90.

[8] Grewal SS, Barker JN, Davies SM, Wagner JE. Unrelated donor

hematopoietic cell transplantation: marrow or umbilical cord

blood? Blood 2003;101:4233–44.

[9] Lee KB, Hui JH, Song IC, Ardany L, Lee EH. Injectable

mesenchymal stem cell therapy for large cartilage defects – a

porcine model. Stem Cells 2007;25:2964–71.

[10] Kuroda R, Usas A, Kubo S, Corsi K, Peng H, Rose T, et al.

Cartilage repair using bone morphogenetic protein 4 and muscle-

derived stem cells. Arthritis Rheum 2006;54:433–42.

[11] Yamamoto T, Wakitani S, Imoto K, Hattori T, Nakaya H, Saito

M, et al. Fibroblast growth factor-2 promotes the repair of

partial thickness defects of articular cartilage in immature rabbits

but not in mature rabbits. Osteoarthritis Cartilage

2004;12:636–41.

[12] Eichler H, Richter E, Leveringhaus A, Zieger W, Watz E,

Friedmann G, et al. The mannheim cord blood project: experi-

ence in collection and processing of the first 880 banked unrelated

cord blood transplants. Infusion Ther Transfus Med

1999;26:110–4.

[13] Mackay AM, Beck SC, Murphy JM, Barry FP, Chichester CO,

Pittenger MF. Chondrogenic differentiation of cultured human

mesenchymal stem cells from marrow. Tissue Eng 1998;4:415–28.

[14] Nielsen H. Isolation and functional activity of human blood

monocytes at adherence to plastic surfaces: comparison of

different detachment methods. Acta Pathol Microbiol Immunol

Scand 1987;95:81–4.

[15] Murphy JM, Heinegard R, McIntosh A, Sterchi D, Barry FP.

Distribution of cartilage molecules in the developing mouse joint.

Matrix Biol 1999;18:487–97.

[16] Joosten LAB, Lubberts E, Helsen MA, Saxne T, Coenen-deRoo

CJ, Heinegard D, et al. Protection against cartilage and bone

destruction by systemic 1L-4 treatment in established murine type

II collagen induced arthritis. Arthritis Res 1999;1:81–91.

[17] Elders MJ. The increasing impact of arthritis on public health. J

Rheumatol Suppl 2000;60:6–8.

[18] Brooks PM. Impact of osteoarthritis on individuals and society:

how much disability? Social consequences and health economic

implications. Curr Opin Rheumatol 2002;14:573–7.

[19] Hochberg MC, Altman RD, Brandt KD, Clark BM, Dieppe PA,

Griffin MR, et al. Guidelines for the medical management of

osteoarthritis. Part II. Osteoarthritis of the knee. American

College of Rheumatology. Arthritis Rheum 1995;38:1541–6.

[20] Gunther KP. Surgical approaches for osteoarthritis. Best Pract

Res Clin Rheumatol 2001;15:627–43.

Please cite this article in press as: Kamel NS et al. Effect of intra-articularanimals, The Egyptian Rheumatologist (2014), http://dx.doi.org/10.1016/j

[21] Moseley JB, O’Malley K, Petersen NJ, Menke TJ, Brody BA,

Kuykendall DH, et al. A controlled trial of arthroscopic surgery

for osteoarthritis of the knee. N Engl J Med 2002;347:81–8.

[22] Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R,

Mosca JD, et al. Multilineage potential of adult human mesen-

chymal stem cells. Science 1999;284:143–7.

[23] Vanwanseele B, Lucchinetti E, Stussi E. The effects of immobi-

lization on the characteristics of articular cartilage: current

concepts and future directions. Osteoarthritis Cartilage

2002;10(5):408–19.

[24] Bendele AM, White SL. Early histopathologic and ultrastructural

alterations in femorotibial joints of partial medial meniscectom-

ized guinea pigs. Vet Pathol 1987;24:436–43.

[25] Yanai T, Ishii T, Chang F, Ochiai N. Repair of large full-

thickness articular cartilage defects in the rabbit; the effects of

joint distraction and autologous bone marrow-derived mesenchy-

mal cell transplantation. J Bone Joint Sug 2005;87(5):721–9.

[26] Cao FJ, Feng SQ. Human umbilical cord mesenchymal stem cells

and the treatment of spinal cord injury. Chin Med J

2009;122(2):225–31.

[27] Erices A, Conget P, Minguell JJ. Mesenchymal progenitor cells in

human umbilical cord blood. Br J Haematol 2000;109:235–42.

[28] Rosada C, Justesen J, Melsvik D, Ebbesen P, Kassem M. The

human umbilical cord blood: a potential source for osteoblast

progenitor cells. Calcif Tissue Int 2003;72:135–42.

[29] Gluckman E. Umbilical cord blood biology and transplantation.

Curr Opin Hematol 1995;2:413–6.

[30] Malek A, Bersinger NA. Human placental stem cells: biomedical

potential and clinical relevance. J Stem Cells 2011;6(2):75–92.

[31] Jackson DW, Simon TM. Tissue engineering principles in

orthopedic surgery. Clin Orthop 1999;367:S31–45.

[32] Solchaga LA, Yoo JU, Lundberg M, Dennis JE, Huibregtse BA,

Goldberg VM, et al. Hyaluronan-based polymers in the treat-

ment of osteochondral defects. J Orthop Res 2000;18:773–80.

[33] Ponticello MS, Schinagl RM, Kadiyala S, Barry FP. Gelatin-

based resorbable sponge as a carrier matrix for human mesen-

chymal stem cells in cartilage regeneration therapy. J Biomed

Mater Res 2000;52:246–55.

[34] Barry FP. Mesenchymal stem cell therapy in joint disease. In:

Bock G, Goode J, editors. Tissue Engineering of Cartilage and

Bone, vol. 249. John Wiley & Sons; 2002. p. 86–96.

[35] Bos BK, Kops N, Verhaar JA, van Osch GJ. Cellular origin of

neocartilage formed at wound edges of articular cartilage in a

tissue culture experiment. Osteoarthritis Cartilage

2008;16(2):204–11.

[36] Yan H, Yu C. Repair of full-thickness cartilage defects with cells

of different origin in a rabbit model. Arthroscopy

2007;23(2):178–87.

[37] Nam HY, Karunanithi P, Loo WCP, Naveen S, Chen H, Hussin

P, et al. The effects if staged intra-articular injection of cultured

autologous mesenchymal stromal cells on the repair of damaged

cartilage: a pilot study in caprine model. Arthritis Res Ther

2013;15:R129.

injection of mesenchymal stem cells in cartilage repair in experimental.ejr.2014.03.001