Evaluation of Articular Cartilage Progenitor Cells for the Repair of ...

484 C OPYRIGHT 2015

BY

T HE J OURNAL

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B ONE

AND J OINT

S URGERY, I NCORPORATED

Evaluation of Articular Cartilage Progenitor Cells for the Repair of Articular Defects in an Equine Model David D. Frisbie, DVM, PhD, Helen E. McCarthy, BSc, MSc, PhD, Charles W. Archer, BSc, PhD, Myra F. Barrett, MS, DVM, and C. Wayne McIlwraith, BVSc, PhD, DSc Investigation performed at the Orthopaedic Research Center, Colorado State University, Fort Collins, Colorado

Background: We sought to determine the effectiveness of chondroprogenitor cells derived from autologous and allogenic articular cartilage for the repair of cartilage defects in an equine model. Methods: Cartilage defects (15 mm) were created on the medial trochlear ridge of the femur. The following experimental treatments were compared with empty-defect controls: fibrin only, autologous chondroprogenitor cells plus fibrin, and allogenic chondroprogenitor cells plus fibrin (n = 4 or 12 per treatment). Horses underwent strenuous exercise throughout the twelve-month study, and evaluations included lameness (pain) and arthroscopic, radiographic, gross, histologic, and immunohistochemical analyses. Results: Arthroscopy and microscopy indicated that defects in the autologous cell group had significantly better repair tissue compared with defects in the fibrin-only and control groups. Repair tissue quality in the allogenic cell group was not superior to that in the fibrin-only group with the exception of the percentage of type-II collagen, which was greater. Radiographic changes in the allogenic cell group were poorer on average than those in the autologous cell group. Autologous cells significantly reduced central osteophyte formation compared with fibrin alone. Conclusions: On the basis of the arthroscopic, radiographic, and histologic scores, autologous cells in fibrin yielded better results than the other treatments; allogenic cells cannot be recommended at this time. Clinical Relevance: Autologous chondroprogenitor cells in fibrin appeared to yield a modest improvement over fibrin alone, with a 128% difference in central osteophyte formation compared with fibrin-only treatment. This cell type may show clinical benefit, and comparisons with other cartilage resurfacing techniques should be considered.

Peer Review: This article was reviewed by the Editor-in-Chief and one Deputy Editor, and it underwent blinded review by two or more outside experts. It was also reviewed by an expert in methodology and statistics. The Deputy Editor reviewed each revision of the article, and it underwent a final review by the Editor-in-Chief prior to publication. Final corrections and clarifications occurred during one or more exchanges between the author(s) and copyeditors.

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ocal chondral defects are identified in more than onehalf of the arthroscopy procedures performed in the human knee1-3, and similar numbers are seen in clinical cases of equine stifle (knee) joint arthroscopy4. Despite a vast amount of research, a recent systematic review of Level-I and II studies involving 421 human patients who had been treated

with autologous chondrocyte implantation, osteochondral autograft transfer, matrix-induced autologous chondrocyte implantation, or microfracture and who had long-term followup indicated that no technique consistently yielded superior results5, confirming that improved cartilage repair techniques are desired.

Disclosure: One or more of the authors received payments or services, either directly or indirectly (i.e., via his or her institution), from a third party in support of an aspect of this work. None of the authors, or their institution(s), have had any financial relationship, in the thirty-six months prior to submission of this work, with any entity in the biomedical arena that could be perceived to influence or have the potential to influence what is written in this work. One or more of the authors has a patent or patents, planned, pending, or issued, that is broadly relevant to the work. No author has had any other relationships, or has engaged in any other activities, that could be perceived to influence or have the potential to influence what is written in this work. The complete Disclosures of Potential Conflicts of Interest submitted by authors are always provided with the online version of the article.

J Bone Joint Surg Am. 2015;97:484-93

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http://dx.doi.org/10.2106/JBJS.N.00404

485 TH E JO U R NA L O F B O N E & JO I N T SU RG E RY J B J S . O RG V O L U M E 97-A N U M B E R 6 M A R C H 18, 2 015 d

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E VA LUAT I O N O F A R T I C U L A R C A R T I L A G E P R O G E N I T O R C E L L S F O R T H E R E PA I R O F A R T I C U L A R D E F E C T S

TABLE I Histologic Outcomes* Parameter and Group

Six Months

Twelve Months

Nature of predominant tissue Auto Allo CNT Fibrin

0.48 0.04 0.94 0.49

± 0.35, a ± 0.43, a ± 0.31, a ± 0.25, a

0.75 0.50 0.00 0.18

± 0.21, a ± 0.21, ab ± 0.21, b ± 0.12, b

Surface regularity Auto Allo CNT Fibrin

1.00 2.00 2.00 1.75

± 0.30, a ± 0.37, a ± 0.26, a ± 0.18, a

2.88 1.63 2.00 2.22

± 0.30, a ± 0.30, b ± 0.30, b ± 0.18, ab

Structural integrity Auto Allo CNT Fibrin

1.33 ± 0.24, a 20.00 ± 0.30, b 0.75 ± 0.21, ab 0.87 ± 0.15, ab

1.38 1.13 0.88 1.27

± 0.21, a ± 0.21, a ± 0.21, a ± 0.12, a

Repair tissue filling Auto Allo CNT Fibrin

1.33 ± 0.24, a 20.00 ± 0.30, b 0.74 ± 0.21, ab 0.87 ± 0.15, ab

1.75 1.25 0.75 1.39

± 0.19, a ± 0.19, ab ± 0.19, b ± 0.11, a

NA NA NA NA

1.88 1.75 2.00 1.78

± 0.17, a ± 0.17, a ± 0.17, a ± 0.10, a

Bonding to adjacent cartilage Auto Allo CNT Fibrin Hypocellularity Auto Allo CNT Fibrin

0.00 0.00 0.00 0.00

± 0.18, a ± 0.18, a ± 0.18, a ± 0.18, a

Chondrocyte clustering Auto Allo CNT Fibrin

2.00 2.00 2.00 2.00

± 0.17, a ± 0.17, a ± 0.17, a ± 0.17, a

1.75 1.75 2.00 1.87

± 0.17, a ± 0.17, a ± 0.17, a ± 0.10, a

NA NA NA NA

2.13 1.88 1.50 1.96

± 0.15, a ± 0.15, ab ± 0.15, b ± 0.10, a

± 0.14, a ± 0.14, a ± 0.14, a ± 0.14, a

2.00 1.75 1.50 1.96

± 0.16, a ± 0.16, ab ± 0.16, b ± 0.10, a

Degenerative change in surrounding articular cartilage Auto Allo CNT Fibrin Reconstitution of subchondral bone Auto Allo CNT Fibrin

2.45 2.45 2.45 2.45

Inflammatory response in subchondral bone region Auto Allo

1.78 ± 0.27, a 1.97 ± 0.33, a

20.00 ± 0.18, b 0.63 ± 0.18, a 20.00 ± 0.18, b 0.14 ± 0.11, b

1.75 ± 0.19, a 1.00 ± 0.19, b

continued


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TABLE I (continued) Parameter and Group CNT Fibrin Safranin O Auto Allo CNT Fibrin Cumulative total histology score Auto Allo CNT Fibrin

Six Months

Twelve Months

1.71 ± 0.23, a 1.76 ± 0.17, a

1.13 ± 0.19, b 1.69 ± 0.11, a

0.39 0.51 0.48 0.86

± 0.27, a ± 0.33, a ± 0.23, a ± 0.17, a

1.75 1.13 1.00 1.35

± 0.15, a ± 0.15, b ± 1.15, b ± 0.10, b

12.77 11.89 12.15 12.84

± 0.98, a ± 1.16, a ± 0.90, a ± 0.78, a

18.00 14.38 12.75 15.80

± 0.92, a ± 0.92, bc ± 0.92, c ± 0.54, b

*Assessed in the six-month biopsy sample or at the twelve-month end point; higher values are better. The values are subjective scores and are given as the mean and the standard error of the mean. Groups labeled with the same letter did not differ significantly from each other. NA = not applicable.

Bone-marrow-derived mesenchymal stem cells (MSCs) implanted in a fibrin-based matrix have not shown long-term benefit6. Furthermore, it is our experience that MSCs implanted in a fibrin matrix or in a matrix with other increased growth factors lead to excessive mineralization of the repair tissue. Our group has characterized a novel cell type, isolated from the superficial layer of both human7 and equine articular cartilage, that may have characteristics superior to those of bone-marrow-derived MSCs for matrix implantation. Equine articular cartilage progenitor cells demonstrate a multipotent differentiation capacity similar to that of bone-marrow-derived MSCs8. Specifically, these cells form colonies from an initial low seeding density and have been shown to express the putative stem cell markers STRO-1, CD90, and CD166. In vitro, chondrogenic induction reveals positive labeling for type-II collagen and aggrecan without detection of type-X collagen. The lack of type-X collagen or a hypertrophic cartilage (endochondral) phenotype is expected to limit repair tissue mineralization in vivo. Unlike mature chondrocytes, these cartilage progenitor cells exhibit delayed senescence and retain their chondrogenic potential following extensive in vitro expansion9. The cells have been shown to demonstrate plasticity both in vitro and in vivo7,8,10 and maintain Sox-9 gene expression in monolayer culture during up to fifty population doublings9,11. The purpose of this study was to evaluate autologous and allogenic cartilage progenitor cells implanted in a fibrin matrix to heal critical-sized articular defects over a twelve-month period in an equine model that included controlled strenuous exercise. Comparisons were made with empty defects and defects filled with fibrin only. Materials and Methods

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our defect treatments were compared: (1) autologous chondroprogenitor cells transplanted in fibrin, (2) allogenic chondroprogenitor cells trans-

planted in fibrin, (3) fibrin alone, and (4) no filling of the defects. The evaluators of each outcome parameter were blinded to the treatment groups and were experts in that particular area.

Animals Twelve skeletally mature horses (age, two to six years) with normal musculoskeletal examination results were utilized in this study, which was approved by our Institutional Animal Care and Use Committee. All procedures (cartilage harvesting, defect creation and construct implantation, six-month biopsy, and twelve-month arthroscopy) were performed under general anesthesia. Afterward, each horse received 4.5 mg/kg of phenylbutazone (Equi-Phar; Vedco) once a day for a total of five days as well as 2.2 mg/kg of ceftiofur sodium (Naxcel; Zoetis) intramuscularly twice a day for three days. Bilateral harvesting of superficial cartilage was performed by means of arthroscopy; approximately 100 mg was harvested from the proximal aspect 12 of the lateral trochlear ridge on each side . Cartilage from one randomly chosen side was utilized for autologous treatment, and cartilage from the contralateral side was utilized for allogenic treatment of a different, randomly chosen horse. Horses were housed in stalls (3.65 · 3.65 m) throughout the study 12 except during exercise sessions (see Appendix) .

Chondroprogenitor Cells Superficial-zone cartilage was digested, filtered, resuspended in 1 mL of serumfree medium consisting of DMEM (Dulbecco modified Eagle medium)/F12 plus 100 mg/mL gentamicin, 0.1 mM ascorbate-2-phosphate, 0.5 mg/mL L-glucose, and 10 mM HEPES (4-[2-hydroxyethyl]-1-piperazineethanesulfonic acid) buffer. A progenitor cell population was isolated according to the method of 10 Dowthwaite et al. . Cells were seeded at 4000 cells/mL in serum-free medium and allowed to grow; nonadherent cells were subsequently removed and adherent cells were cultured in growth medium consisting of serum-free medium plus 10% FBS (fetal bovine serum). On day 6, colonies with more 13 than thirty-two cells were marked for cloning , isolated, trypsinized, and transferred into 12-well plates containing growth medium with 1 ng/mL of TGF (transforming growth factor)-b1 and 5 ng/mL of FGF (fibroblast growth factor)-2. After fourteen days of expansion, cells were cryopreserved in liquid nitrogen.


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TABLE II Immunohistochemical Outcomes Protein and Group

Percentage*

Type-I collagen Auto

40.83 ± 14.82, a

Allo CNT

59.17 ± 14.82, a 64.33 ± 14.82, a

Fibrin

49.89 ± 8.34, a

Type-X collagen Auto

4.50 ± 1.13, a

Allo

2.00 ± 1.13, a

CNT

1.88 ± 1.13, a

Fibrin

3.83 ± 0.65, a

Aggrecan Auto

76.04 ± 13.03, a

Allo CNT

47.50 ± 14.77, a 41.04 ± 13.03, a

Fibrin

63.25 ± 7.63, a

Type-II collagen Auto

46.25 ± 7.34, a

Allo

37.50 ± 7.34, a

CNT

6.88 ± 7.34, b

Fibrin

17.54 ± 4.24, b

*Percentage of repair tissue staining positive for the corresponding antibody compared with the negative control. The values are given as the mean and the standard error of the mean. Groups labeled with the same letter did not differ significantly from each other.

Viability after thawing was assessed by a trypan blue assay and was found to be 88% or greater. Cell numbers used at implantation were adjusted to correct for nonviable cells. A 50% autologous fibrinogen solution was created by diluting fibrinogen in sterile PBS (phosphatebuffered saline solution). Chondroprogenitor cells were mixed with this solution at a concentration of 30 million cells/mL (36 million viable cells in 600 mL of fibrinogen solution and 600 mL of thrombin); a cell-free fibrinogen solution was used for defects that were to be filled with fibrin only.

Defect Creation and Filling A defect 15 mm in diameter and extending down to the level of the subchondral bone plate was made on the medial aspect of the trochlea at the distal aspect 12 of each femur through a femoropatellar arthrotomy . The dry defect was slightly overfilled with the autologous fibrin and thrombin solution (1:1 ratio by volume) with or without cells or was left empty. One defect in each animal was filled with fibrin alone (Fibrin group, n = 12). The defect in the contralateral limb received autologous cells in fibrin (Auto group, n = 4), received allogenic cells in fibrin (Allo group, n = 4), or remained empty (CNT [control] group, n = 4).

Postoperative Evaluation Second-look arthroscopy, performed at six and twelve months after defect creation, assessed repair tissue attachment to surrounding articular cartilage and bone, the presence of acute or chronic hemorrhage, degeneration of defect

margins, firmness, level or filling, and the overall grade of repair tissue . Criteria were assessed on a scale of 0 to 4, with 0 being normal and 4 being a severe change. At six months, a 4-mm osteochondral biopsy sample encompassing 2 mm of repair tissue and 2 mm of surrounding articular cartilage was harvested; the twelve-month assessment was carried out as part of a terminal surgical procedure. Musculoskeletal examinations were performed at two-month intervals after defect creation and included four different outcomes: (1) the American Association of Equine Practitioners scale for the degree of pain (on a scale of 0 to 5, with 0 being normal and 5 being non-weight-bearing), (2) the degree of synovial effusion, (3) the pain response after flexion of the affected joint (on a scale of 0 to 4, with 0 being normal to 4 being severe pain), and (4) the range of motion of the femorotibial joint (on a scale of 0 to 3, with 0 being normal and 3 being severely reduced). Femoropatellar radiographs were obtained at two-month intervals. Outcome parameters included osseous proliferation adjacent to the defect (central osteophytes), lysis, sclerosis, peripheral osteophyte formation, and filling of the defect. Criteria were graded from 0 to 4, with 0 being normal and 4 being severe except in the case of radiographically evident filling, where 0 was none, 1 was 1% to 25%, 2 was 26% to 50%, 3 was 51% to 75%, and 4 was 76% to 100% filling of the originally created defect. Gross assessment of the medial trochlear ridge was performed after the twelve-month arthroscopy and euthanization of the animals. This assessment involved repair tissue attachment to surrounding articular cartilage and bone; degeneration of defect margins and repair tissue; the firmness, area, and volume of repair tissue; and the overall grade of repair tissue on the basis of all parameters. Criteria were graded on a scale of 0 to 4, with 0 being normal and 4 being severe. Histologic examinations were performed on the six-month biopsy sample as well as two osteochondral samples per defect (one in the proximal third and one in the distal third of the defect) at twelve months; these were processed and graded in a similar fashion. After formalin fixation and decalcification using dilute formic acid, samples were embedded in paraffin, sectioned, and stained with hematoxylin and eosin or with safranin O and fast green. Each section was graded on the basis of criteria outlined in the Ap14,15 pendix . Synovial membrane was also collected at twelve months. After formalin fixation, these samples were embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Each synovial outcome parameter (cellular infiltration, intimal hyperplasia, subintimal edema, subintimal fibrosis, and vascularity) was graded on a scale of 0 to 4, with 0 being normal and 4 being severe. Immunohistochemical analysis was performed on two samples harvested from each defect at twelve months. These samples, which contained both repair tissue and surrounding cartilage, were embedded in paraffin or frozen embedded in CryoPrep (American Master Tech, Lodi, California). The frozen section was probed to detect type-I collagen (Accurate Chemical & Scientific, Westbury, New York) and aggrecan (Acris Antibodies, San Diego, California). The paraffin section was probed to detect type-II collagen (Developmental Studies Hybridoma Bank, Iowa City, Iowa) and type-X collagen. Control sections not exposed to the primary antibody were probed with normal serum specific to the corresponding primary anti8,12 body . The percentage of the area of the repair tissue that exhibited positive staining (as determined by comparison with the control) was subjectively assessed.

Statistical Analysis Analysis of each outcome parameter was performed with use of SAS software (version 9.2; SAS Institute, Cary, North Carolina) and involved descriptive statistics, nonparametric frequency tables, and/or mixed-model ANOVA (analysis of variance). Mixed-model ANOVA was utilized for continuous data, with treatment and time point as main or interaction variables. A least square means procedure was then utilized to make individual comparisons. Both the Fisher exact test and mixed-model ANOVA were utilized for ordinal data. A p value of 20% (which we considered the threshold for clinical significance) for the arthroscopic grade of the repair tissue, and it improved the amount of repair tissue staining positive for type-II collagen and decreased central osteophyte formation. On the basis of clinical and past in vivo experience that subchondral bone microfracture provided better histologic repair tissue, combining microfracture with autologous use of cartilage progenitor cells may yield an improved longterm repair. This combination may also improve the central osteophyte formation seen with microfracture alone22. The combination of microfracture and autologous progenitor cells may be accomplished by microfracturing the defect and then either adding autologous progenitor cells in fibrin to the defect or injecting the cells free in the joint. The latter has been suc-


cessful in augmenting the results seen with microfracture alone23, and both warrant further investigation. Appendix Tables showing the exercise protocol and histologic grading criteria as well as figures showing the range of flexion and the percentage of repair tissue staining positive for type-II collagen in each treatment group are available with the online version of this article as a data supplement at jbjs.org. n

David D. Frisbie, DVM, PhD Myra F. Barrett, MS, DVM C. Wayne McIlwraith, BVSc, PhD, DSc Orthopaedic Research Center, Department of Clinical Sciences (D.D.F. and C.W.M.), and Department of Radiological and Health Sciences (M.F.B.), College of Veterinary Medicine and Biomedical Sciences, Colorado State University, 300 West Drake Road, Fort Collins, CO 80523. E-mail address for D.D. Frisbie: [email protected] Helen E. McCarthy, BSc, MSc, PhD Division of Pathophysiology and Repair, Cardiff School of Biosciences, Cardiff University, Sir Martin Evans Building, Museum Avenue, Cardiff, CF10 3AX, United Kingdom Charles W. Archer, BSc, PhD Institute of Life Sciences, Swansea University, Singleton Park, Swansea, SA2 8PP, United Kingdom

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