Bone marrow derived mesenchymal stem cell (BM MSC) application in articular cartilage repair 10

Although, these factors may make a difference between behavior of stem cells and injured tissue in vivo and in vitro microfluidic device, microfluidic device has many advantages to the

Figure 4-10 Migration quantification of the MSCs toward uninjured and injured cartilage tissue

Migration distance of the MSCs toward the complete media (CM) alone, uninjured and injured tissues were showed in this graph (Error bars represent standard errors of the means, one star: p-value < 0.05, two star: p-value < 0.01, and star: p-value < 0.001)

4.4.6 Quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR)

Custom-designed RT-PCR array was used to evaluate the differences in gene expressions of uninjured and injured cartilage tissue The factors were chosen according to the literature review and the availability of the primers for the RT-PCR (Table 4.1) RT-PCR results showed that the injured cartilage up-

regulated expressions of collagen type I A1 (COL1A1), chemokine C-X-C motif 10 (CXCL10), transforming growth factor alpha (TGFA), insulin-like growth factor 2 (IGF2), chemokine C-X-C motif 12 (CXCL12), angiopoietin 1 (ANGPT1), fibroblast growth factor 2 (FGF2), transforming growth factor beta-

3 (TGFβ3), bone morphogenetic protein 4 (BMP4), and vitronectin (VTN) ligands Figure 4.8 showed the relative increase of gene expressions of these factors

Figure 4-11 Gene expression change of candidate ligands in injured cartilage

Gene expression level (sample number = 2) increases of the candidate

ligands, which could be involved in the MSCs migration

4.5 Discussion

To evaluate if the migration of MSCs toward the injured cartilage, which was shown in previous chapter, is an active reaction to the injury site or a random translocation of MSCs to that site, the behavior of MSCs were studied in a

microfluidic device I developed a new microfluidic device to simulate the in vivo interaction of MSCs with injured cartilage tissue in a three dimensional

(3D) environment This simulation allows monitoring of the interactions

between MSCs and the tissue in cellular and molecular level However, our

system still lacks many of the characteristics of an actual in vivo situation For

example in living animals, the host cells such as immune cells (e.g

macrophages, monocytes, T cells, etc.) have interactions with both injured tissues and transplanted stem cells, which may affect their behaviors

Moreover, in in vivo the blood circulation and joint motion may have some role

in stem cell migration, which I do not have in this in vitro model Although,

these factors may make a difference between behavior of stem cells and

injured tissue in vivo and in vitro (microfluidic device), microfluidic device has

many advantages to the current methods and mimics the environment of real tissue better than conventional methods such as Boyden chamber, scratch migration assay, and under agarose migration system

Boyden chamber, scratch test and under-agarose migration assay are the most common migration assay systems The Boyden chamber assay uses a membrane to separate the upper chamber (cell chamber) from the bottom chamber, which is too thin to make a gradient In Boyden chamber assay only the final results of the migration can be collected and this system allows testing only one condition at a time In the scratch migration test, cells are

scratched and the migration of the cells is evaluated by monitoring how the cells fill the gap.This system does not make a gradient of the chemotactic factors, which is one of the effective components in the tissue regeneration Also with this system we cannot use any tissue as the origin of chemotactic factors Under agarose migration assay system is dependent on the concept that solidified agarose does not attach to glass surfaces To perform this assay, a thick layer of warm agarose should be poured into a glass plate After solidification, three holes punched out; one hole for cell seeding, one for the cytokine source and one as control After gradient formation of the

cytokine the cell migration can be observed, however this migration can be in any direction, and monitoring of the cells can be very difficult Other potential limitation of this system is the risk of cross-contamination of the cytokines between the holes through the porous agarose

By using microfluidic system, I could study the cell migration in a 3D

environment, which was more similar to in vivo situation and provided the

control of the gradient between channels By having the specific channels for the 3D scaffold parallel to the cell channel on both sides, I decreased the risk

of cross-contamination of the cytokines and also I could control the migration

of the cells in a certain direction (collagen channel), which made it easier to monitor and image the cells migration Moreover, the high quality imaging capabilities of microfluidic system provided real-time monitoring of cells

simultaneously at two different conditions over time

The results of this study showed that MSCs could be primed and migrated toward the injured cartilage The migration (distance) toward the injured tissue

is longer than that of uninjured cartilage, suggesting injured tissue may secret factors attracting the MSCs

As I showed that the injured cartilage attracts the MSCs, and the engraftment

of the MSCs in the injured cartilage could be an active migration and homing,

I also evaluated the potential chemotactic candidates for this phenomenon There are many chemotactic factors named in the literature that are secreted

by different injured tissues such as skin wound, acute and chronic

inflammation in brain and etc Previous studies (Table 4-2) have shown that CXCL10 (247), TGFA (157), IGF2 (152), CXCL12 (248), FGF2 (148), TGFB3 (249), BMP4 (154) and ANGPT1 (158) are stimulatory factors for MSCs migration However, to our knowledge, there is not any study on the injured cartilage As the nature of the cartilage is different from the other tissues due

to lack of blood vessels and lymphatic drainage, in this study I evaluated the factors, which were up-regulated by the chondrocytes after acute cartilage injury It is crucial to understand the chemotactic factors secreted by injured cartilage to be able to use a sub-population of MSCs, which show stronger response to such factors in the clinical setting to design more effective

treatment plan for patients

RT-PCR results of injured cartilage tissues demonstrated that, chemotactic factors such as CXCL10, TGFA, IGF2, CXCL12, ANGPT1, FGF2, TGFB3, BMP4, and the extracellular matrix (ECM) proteins genes such as COL1A1, and VTN were up-regulated after cartilage injury

Table 4-2 Other studies done on stem cell stimulatory chemotactic factors

Ligand

COL1A1 Commercially available Modified Boyden chamber Collagen I induced significant motogenic activity for both rabbit and human MSCs Thibault et al (161) CXCL10 Recombinant human chemokine Agarose drop migration assay CXCL10 chemokine trigger hMSC migration and promote hMSC proliferation Rice et al (247) TGFA Commercially available Boyden chamber / Wound assay The factors that induced the migration of rabbit and human MSCs also enhanced their proliferation Ozaki et al (157) IGF2 Recombinant human chemokine Modified Boyden chamber

IGF2 is a chemotactic factor for hMSCs and stimulates migration of human mesenchymal progenitor cells

Fiedler et al (152) CXCL12

Supernatant of cultured human pancreatic islets

Modified Boyden chamber

Human pancreatic islets as an in vitro model released CXCL12, which is able to attract BM MSCs in vitro

Sordi et al (248)

Migration values of the TNFα-stimulated BM MSCs were higher than un-stimulated cells

Ponte et al (158)

available

Boyden chamber / Wound Assay / methyl cellulose disc

Low concentrations of FGF2 leads to migration, whereas higher concentrations resulted in repulsion

of the MSCs

Schmidt et al (148) TGFB3 Commercially available Modified Boyden chamber TGFB3 stimulates chemotaxis/chemokinesis of multipotent C3H10T1/2 cells Makhijani et al (249)

available

Modified Boyden chamber

Migration of primary human progenitor cells was stimulated by rxBMP-4 in a dose-dependent manner

Fiedler et al (154) VTN Commercially available Modified Boyden chamber Vitronectin induced significant motogenic activity for both rabbit and human MSCs Thibault et al (161)

Our results agreed with those of Thibault et al who demonstrated that ECM proteins such as Col1 and VTN could induce significant migratory and

motogenic activity for MSCs (161) Then, these ECM proteins could be used

in the clinical setting for cartilage repair as a scaffold to carry the stem cells and/or attract the endogenous or exogenous stem cells (endogenous from the bone marrow and exogenous by multiple intra-articular injection of the

expanded autologous stem cells)

As I showed, in the previous chapter, that injection of stem cells is a promising method for cartilage repair, in this chapter I confirmed that engraftment of the MSCs in injured cartilage is an active migration and homing process and injured cartilage encourage the migration of the MSCs toward the injury site I also showed that the cartilage injury up-regulate some specific chemotactic factors, which can help to find and select a sub-population of MSCs which show stronger response to such factors in cartilage repair On one hand, enhancement of the homing capacity of MSC can be achieved by modulating their response to chemotactic factors; for example by finding and selecting sub-population of MSCs which show stronger response to such factors

(because of higher expression of surface receptors which responsible for those chemotactic signals) (250) On the other hand, modulation can be

applied in the site of injury for example with stimulating the target site to

attract more MSCs (with releasing more signals)

Chapter 5 Autologous Bone Marrow

Derived Mesenchymal Stem Cell versus Autologous Chondrocyte Implantation: An Observational Cohort Study 1

1 The final, definitive version of this paper has been published in “The

American Journal of Sports Medicine”, 38(6): 1110-6, 2010 June by SAGE Publications Ltd SAGE Publications, Inc., All rights reserved ©

5.1 Abstract

Background: First generation ACI has limitations and introducing new

effective cell sources can improve cartilage repair

Purpose: To compare the clinical outcomes of patients treated with first

generation autologous chondrocyte implantation (ACI) to patients treated with autologous bone marrow derived mesenchymal stem cell (BM MSCs)

Study Design: Cohort Study, Level of Evidence, 3

Methods: Seventy-two matched (lesion site and age) patients underwent

cartilage repair using chondrocytes (n=36) or BM MSCs (n=36) Clinical outcomes were measured pre-operation and 3, 6, 9, 12, 18, and 24 months post-operation using the International Cartilage Repair Society (ICRS)

Cartilage Injury Evaluation Package which included questions from the Form (SF-36) Health Survey, International Knee Documentation Committee (IKDC) subjective knee evaluation form, Lysholm24 knee scale, and Tegner activity level scale

Short-Results: There was significant improvement in the patients’ quality of life

(physical and mental components of the SF-36 questionnaire included in the ICRS package) after cartilage repair in both groups (ACI and BM MSCs) However, there was no difference between the BM MSCs and the ACI group

in terms of clinical outcomes except for “Physical Role Functioning” with a

greater improvement over time in the BM MSCs group (P = 0.044 for

interaction effect) IKDC subjective knee evaluation (P = 0.861), Lysholm (P = 0.627), and Tegner (P = 0.200) scores did not have any significant difference

between groups over time However, in general, men showed significantly better improvements than women Patients younger than 45 years scored

significantly better than patients older than 45 years in the ACI group; but age did not make a difference in outcomes in the BM MSCs group

Conclusion: Using BM MSCs in cartilage repair is as effective as

chondrocytes for articular cartilage repair In addition, it required one less knee surgery, reduced costs, and minimized donor site morbidity

Key Terms: chondrocyte; autologous chondrocyte implantation (ACI); bone

marrow derived mesenchymal stem cell

5.2 Introduction

Full-thickness, focal cartilage defects causes knee symptoms such as pain, popping and swelling (215); and it affects patient's quality of life and career Recent large arthroscopic studies indicated that the prevalence of cartilage defects is between 11% to 63% (251-253) Treatment of articular cartilage defects remains challenging (254-256), because cartilage tissue has a limited capacity for repair (212, 213, 257) One of the most promising treatments for cartilage defects is Autologous Chondrocyte Implantation (ACI) (29, 258-260), which provides durable, hyaline-like cartilage (261, 262) ACI has some

limitations such as need for general anesthesia or at least regional anesthesia

to harvest the cartilage biopsy, a slow rate of chondrocyte proliferation,

difficulty in obtaining adequate number of chondrocytes for implantation, and

donor site morbidity Some of these limitations could be solved by using other techniques such as second or third generation ACI (263, 264), arthroscopic second generation ACI, and microfracture (265, 266), or introducing new cell sources like debrided waste chondrocytes, Bone Marrow derived

Mesenchymal Stem Cells (BM MSCs), or any combination of these cells (29,

214, 215, 267, 268)

Various authors have suggested the use of BM MSCs for cell-based cartilage repair (51, 61, 214, 215, 269); Bone Marrow-derived Mesenchymal Stem Cells (BM MSCs) have a better proliferation rate than chondrocytes and have differentiation capacity to different tissues including chondrogenesis (270-272) We also showed in the previous chapters that injured cartilage could attract the BM MSCs to home and engraft in the cartilage defect and increase the cartilage repair quality However, as far as we know, cartilage repair by

using BM MSCs has not been compared with other cell sources Then in this chapter, we compared the clinical outcomes of cartilage repair in patients treated by autologous BM MSCs and chondrocytes

As this was a clinical study, surgeries were done by my supervisor (A/prof James Hui) and I assisted him in some of the surgeries I designed the study

as historical cohort study and I used ACI treated patients’ data archive and current data from the BM MSCs implanted patients After I collected the data,

by consulting an independent biostatistician, I analyzed the data Then I interpreted the results and prepared the peer-reviewed manuscript

5.3 Methods

5.3.1 Participants

This non-randomized observational cohort study was designed to investigate the effectiveness of Chondrocytes and BM MSCs as cell sources for repairing full-thickness cartilage defects of the knee The inclusion criteria were, at least, one symptomatic chondral lesion diagnosed by clinical examination and magnetic resonance imaging (MRI) on the femoral condyle, trochlea, or

patella and non-existent or correctable concomitant pathologies The

exclusion criteria were patients with inflammatory arthritis, tri-compartmental osteoarthritis, limited range of motion in particular fixed flexion deformity and those who were 65 years of age or older Cartilage repair was conducted with informed consent of the patients

Patients who fulfilled the inclusion and exclusion criteria were treated by our senior author (JH Hui) Thirty-six consecutive patients underwent BM MSCs and were matched by 36 cases of ACI performed earlier, in terms of lesion sites and (10-year) age intervals

The study protocol was approved by the National Healthcare Group Specific Review Board (NHG DSRB reference number D/00/814) and the University Hospital Ethic Committee In addition, cells were processed at the GMP cell processing facility at the National University Hospital of Singapore

Domain-5.3.2 Cell Sources

5.3.2.1 Chondrocyte (ACI) preparation

The chondrocyte preparation method was adopted from Brittberg et al (29) as described here A small amount of cartilage tissue (1cm x 0.5cm) was taken

from non-weight bearing areas, which were deemed macroscopically healthy

by arthroscopy The harvested tissue was transferred into a specimen

container filled with sterile saline (about 10ml) and processed within 60

minutes The sample was washed twice with PBS (Gibco BRL, Grand Island,

NY, US) and then minced prior to being transferred aseptically into a tube with 5ml collagenase NB6 (Sigma, St Louis, Missouri, US) for overnight digestion

at 37°C in a water bath Digested chondrocytes were washed with DMEM/F12 (Gibco BRL, Grand Island, NY, US) supplemented with 10% FBS (Gibco BRL, Grand Island, NY, US) to stop the enzymatic reaction These cells were then cultured in T75cm2 flasks with DMEM/F12 containing 10% FBS (Gibco) and 50µg/ml L-Ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma,

St Louis, Missouri, US) in a humidified atmosphere of 5% CO2, 37°C Cells were seeded at a cell density of 5,000 cells per square centimeter Initial medium change was done after 7 days, when adherent cells were recognized Subsequent medium change was done two to three times a week until the preparation of cell sheets, which were formed in the presence of Ascorbic acid (Passage 1) For each surgery, at least 4 cell sheets were prepared and around two million cells /cm2 were applied

5.3.2.2 MSCs preparation

The detailed method is as follows; Under local anesthesia, 30 ml of bone marrow (BM) was aspirated using a Jamshidi needle from the iliac crests of each patient into heparinized syringes and transferred into sterile containers Seventy or eighty milliliters of each patient’s blood were collected as well The bone marrow aspirate was processed within 60 minutes The heparinized bone marrow aspirate was mixed with a one-fifth volume of 6% (w/v) dextran

(molecular weight 100,000; Sigma, St Louis, Missouri, US) and left standing at room temperature for 30 minutes to eliminate erythrocytes The remaining cells were washed twice with DMEM (Gibco BRL, Grand Island, NY, US) These cells were cultured in T75cm2 flasks with an initial culture medium consisting of DMEM (Gibco) containing 10% FBS (Gibco BRL, Grand Island,

NY, US), 50µg/ml L-Ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma, St Louis, Missouri, US) and 1% antibiotic-antimycotic (penicillin

100U/ml, streptomycin 0.1mg/ml, amphotericin B 0.25µg/ml) (Sigma, St Louis, Missouri, US) in a humidified atmosphere of 5% CO2, at 37°C The cells were seeded at a density of 10,000 cells per square centimeter Initial medium change was done after 5 days when adherent cells were recognized

Subsequently, culture media without antibiotics were used and changed two

to three times a week Cell sheets were formed in the presence of Ascorbic acid (Passage 1) and for each surgery, at least 4 cell sheets were prepared and around two million cells /cm2 (which is determined experimentally) were applied This MSC preparation method is a modified approach from Wakitani

et al study (61) In this method we harvested the bone marrow and expanded the cells the same as Wakitani’s approach however, we prepared the cell sheets (by useing Ascorbic acid) instead of cell suspension

Seventy milliliters of venous blood from each patient was transferred into two 50ml tubes for overnight incubation at 4 degree Celsius After centrifuging the tube with slow acceleration, the serum was carefully aspirated and transferred

to a new tube Repeated centrifugation with slow acceleration for 3 minutes at

3000 rpm at ambient temperature was performed The serum was aspirated into a syringe and filtered with a sterile 0.2µm filter The filtered serum was

tested for sterility, anti-HIV and Hepatitis B antigen, and then stored at a temperature of -20 degree Celsius

Flow cytometry against CD90+, CD105+, CD14- and CD34- was used to

confirm that cultured cells were mesenchymal stem cells Saline that was used for transporting the cartilage biopsy to the laboratory, aspirated bone marrow and culture media (without antibiotic) was tested for sterility and

Mycoplasma hominis contamination

5.3.3 Operation techniques

Four to five weeks after harvesting cells, ACI surgery was done For details on ACI, refer to procedure described previously (29) In summary, approximately

10 to 15 million cells (with a viability rate of 96%) were returned for

implantation The cell sheets were transported to the operating theater in a sterile container within the patients’ own serum The debrided chondral defect (without damaging subchondral bone) was measured after arthrotomy

Subsequently, periosteal patch harvesting from the proximal part of the tibia

or distal part of femur was done according to the measured size Next, the harvested periosteum was sutured precisely to the rim of the debrided

defect(s) The cultured chondrocytes or BM MSCs were implanted beneath the patch and very fine stitches (micro suture 7-0) were used to hold the periosteum to the defected site To avoid cell leakage fibrin glue was used to create a watertight seal

5.3.4 Rehabilitation

To derive maximum benefit from the surgery, patients were advised to strictly follow the rehabilitation protocol, which is one of the most important parts of

recovery The rehabilitation protocol began on the day of surgery and includes passive range of motion and isometric muscle contractions Patients were able to begin active motion and partial weight bearing at 6 weeks, progressing

to full weight bearing exercises The rehabilitation protocol varies according to the location and size of the lesion, concomitant procedures, patient’s age and previous activity level There are four areas that rehabilitation focuses on: walking/weight bearing, range of motion, strength, and cardiovascular

capacity

5.3.5 Post operation evaluation

Patients were evaluated preoperatively (pre-operative assessment) as well as

at 3, 6, 9, 12, 18 and 24 months post-operatively Assessments were

performed by our trained research staff using International Cartilage Repair Society (ICRS) Cartilage Injury Evaluation Package which included questions from the Short-Form (SF-36) (273) Health Survey, International Knee

Documentation Committee (IKDC) subjective knee evaluation form, Lysholm knee scale (274), and Tegner activity level scale (275)

Second look arthroscopy was performed in 7 patients (4 in BM MSCs and 3 in ACI group) 9 to 12 months after implantation A biopsy of the repair tissue was obtained in 2 cases (1 in each group) After fixation, paraffin sections were stained with Alcian blue to evaluate aggrecan content and

immunohistochemistry staining was done to assess the collagen type I, II, X content

5.3.6 Statistical analysis

Statistical analysis was performed by consulting an independent

biostatistician using STATA statistical software (Version 10) The MIXED effect model (with random intercept) was used to evaluate the effect of cell-type and time on the quality of life and other functional or pain outcomes of patients, depending on gender This method of analysis appropriately

accounts for the possible correlation between repeated measurements of an individual All statistical evaluations were made, based on an assumption of a two-sided test at the conventional 5% level of significance

5.4 Results

Seventy-two patients who fulfilled the inclusion and exclusion criteria were treated using ACI (n = 36) and BM MSCs (n = 36) between 2001-2005 and 2005-2007 respectively All patients were followed up for 2 years Table 5.1 shows the demographic characteristics of the patients As anticipated,

patients in the two groups had similar age and gender distributions since they were matched by age and gender There were equal numbers of males and females in the ACI group, with a mean age of 42.5 (SD 11.2) years

Correspondingly, in the BM MSCs group, there were 20 men and 16 women with a mean age of 44.0 (SD 11.4) years However, the mean defect sizes of ACI and BM MSCs group were 3.6 cm2 (SD 2.84) and 4.6 cm2 (SD 3.53)

respectively (P-value = 0.270) Concomitant procedures included patella

realignment (6 cases in ACI and 5 cases in BM MSCs group), high tibial osteotomy (5 cases in BM MSCs group), partial meniscectomy (1 in each group), and anterior cruciate ligament reconstruction (1 in each group)

Table 5-1 Demographic characteristics of study patients

Characteristic (n = 36) ACI ABMSCI (n = 36)

Note: Figures in parenthesis denotes percentages unless otherwise indicated.

5.4.1 ICRS package SF-36 components clinical outcomes

Table 5.2 shows the physical and mental components of the SF-36

questionnaire included in the ICRS package Generally, there was a

significant improvement in these quality of life outcomes after ACI over time However, there were no differences between patients treated with BM MSCs

and Chondrocytes in terms of these clinical outcomes (p > 0.05) except for

“Physical Role Functioning” which suggested greater improvements in for patients treated with BM MSCs as compared to chondrocytes, for both males

and females (p value = 0.044 for interaction effect)

Table 5-2 Effects of cell type, time and gender on ICRS package SF-36 component outcomes

* The estimate of the average level of each parameter

** Evaluation of differences between patients treated with BM MSCs or chondrocytes for each parameter

*** Evaluation of the differences over time for each parameter

**** Evaluation of the differences between male and female for each parameter

Within groups, there were significant differences in outcomes between

genders In particular, males demonstrated greater improvements in scores for “Physical functioning”, “Physical Health Summary”, “Physical Role

Functioning”, and “Mental Health Summary” (p < 0.05) On the other hand, gender did not have any effect on the “Vitality”, “Social Functioning”,

Emotional Role Functioning”, “Mental Health”, and “Bodily Pain”

However, there were no differences in physical and mental component scores between the age groups (<45 years versus >= 45 years) within each cell type

Table 5.3 Effect of cell type, time and gender on IKDC, Lysholm, and Tegner outcomes

* The estimate of the average level of each parameter

** Evaluation of differences between patients treated with BM MSCs or chondrocytes for each parameter

*** Evaluation of the differences over time for each parameter

**** Evaluation of the differences between male and female for each parameter.

5.4.2 IKDC subjective knee evaluation outcomes

The postoperative IKDC scores (figure 5.1A) indicated a significant

improvement in performance over time throughout the follow-up period

Patients treated with chondrocytes and BM MSCs did not differ significantly

with regards to the improvements in the IKDC subjective knee evaluation However, men showed significantly better improvements than women (Table

5.3) (P value = 0.022) Moreover, there were no differences in IKDC scores

between patients younger than 45 years and those who were at least 45

years within ACI group (P value = 0.070) and within BM MSCs group (P value

= 0.671)

Figure 5-1 IKDC, Tegner, and Lysholm activity level outcome

5.4.3 Tegner activity level outcomes

The sport activity level was evaluated by the Tegner score As illustrated in figure 5.1B and Table 5.3, there was notable improvement in the Tegner

score during the two-year follow up period (P value < 0.001), and the scores

do not differ between the two cell type groups (P value = 0.200) However, men had more improvement in sports activity than women (P value = 0.011)

Although there was a significant difference in Tegner scores between patients who were ≥ 45 years and those who were younger than 45 years amongst

those treated by ACI (P value = 0.038), no evidence of difference was noted

in the BM MSCs group (P value = 0.307)

5.4.4 Lysholm knee scale outcomes

Lysholm score (figure 5.1C) showed significant improvement over time

However, there was no difference in scores between BM MSCs and ACI groups As described for Tegner and IKDC scores, men had more

improvement than women (Table 5.3) Younger patients (< 45 years) in the

ACI group had better outcomes (P value = 0.010); however, there was no difference between age groups for those treated with BM MSCs (P value

=0.459)

Figure 5-2 Femoral condyle cartilage defect

Femoral condyle cartilage defect after debride- ment (A) Second-look

arthroscopic evaluation 1 year after autologous bone marrow–derived

mesenchymal stem cell implantation (B)

5.4.5 Second-look arthroscopy and histological outcomes

Of the 72 patients, 7 patients (4 in BM MSCs and 3 in ACI group) underwent second look arthroscopy (figure 5.2) as a result of removal of realignment screw, or symptoms of pain, and swelling The surface of the repaired

cartilage was smooth in 3 BM MSCs cases and 2 ACI cases One case in each group had some irregularities There were no evidences of abnormal calcification or necrosis in either group Histological evaluation of the biopsies taken from patients showed hyaline-like cartilage tissue (figure 5.3)

Figure 5-3 Histologic evaluation of biopsy specimens

The biopsy specimens were taken 1 year after autologous bone derives mesenchymal stem cell implantation of femoral condyle

marrow-Immunohistochemistry (IHC) staining against collagen I (A); IHC against collagen II (B); IHC against collagen X (C); and Alcian blue staining against aggrecan (D)

5.5 Discussion

The objective of this study was to compare the effectiveness of BM MSCs to chondrocytes as a cell source for treatment of symptomatic full thickness articular cartilages defects

The strengths of this study were (1) selection of patients according to

established inclusion and exclusion criteria, (2) performing the surgeries by a single surgeon, (3) using validated knee cartilage outcomes instruments, (4) using matched data to decrease the confounding effect of site and age (5) using the same outcome evaluation scales from baseline and different time points, and (6) using a trained independent observer for data collection

In many comparative studies, ACI has been found to be a promising method for cartilage repair (29, 258-261) Fu et al reported significantly greater

improvement in function and pain relief in patients treated with ACI compared

to debridement of cartilage defects in the knee Bentley et al (258)

demonstrated better results for cartilage repair with ACI compared with

mosaicplasty Saris et al (276) and Robinson et al (277) found that ACI produced significantly better repair tissue compared to microfracture

However, Knutsen at al (278, 279) reported similar clinical outcomes

Moreover, new ACI techniques like matrix-associated autologous chondrocyte transplantation/implantation (MACT/MACI) (263, 264), by using biomaterials seeded with chondrocytes as a scaffold instead of a periosteal patch, leads to less surgical time, morbidity and periosteal patch hypertrophy

Another promising method is articular cartilage paste grafting technique, which was developed by Stone et al (280) They used the natural cartilage from the inter-condylar notch and crushed into small pieces and impacted the