WO2002095399A2

WO2002095399A2 - Method for certifying chondrocytes for use in cartilage regeneration

Info

Publication number: WO2002095399A2
Application number: PCT/IB2002/002752
Authority: WO
Inventors: Ming Hao Dr. Zheng; Jiake Dr. Xu
Original assignee: Verigen Ag
Priority date: 2001-03-30
Filing date: 2002-03-29
Publication date: 2002-11-28
Also published as: AU2002339601A1; WO2002095399A3

Abstract

The present invention provides a method for certifying cells for use in cartilage regeneration, the method comprising collecting data indicating chondrocyte cell viability for use in cartilage regeneration and providing a certificate of chondrocyte cell viability including the collecting data. A kit for quality assurance including instructions for collecting data indicating chondroycte cell viability for use in cartilage regeneration and a certificate of chondrocyte cell viability is also included in the present invention. In addition, a method for determining the likelihood of cartilage regeneration by assessing percent apoptosis in a chondrocyte cell culture is also provided.

Description

Method For Establishing Certification Of Chondrocytes

FIELD OF THE INVENTION

The present invention relates to methods for certifying cells and monitoring the quality of chondrocytes for use in cartilage regeneration. It further relates to the detection of cell differentiation and apoptosis of chrondrocytes and determining the viability of chondrocytes, for autologous chondrocyte implantation (ACI).

CROSS REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 60/280,242, filed March 30, 2001, the entire content of which is incorporated herein by reference.

BACKGROUND OF THE INVENTION The articular ends of long bones comprise hyaline cartilage which has a limited capacity for self-repair. The biomechanical integrity of hyaline cartilage is determined by the basic structural compounds of the tissue including chondrocytes, collagen extracellular matrix proteoglycans, non-collagenous proteins and water. Chondrocytes are cells responsible for the production of type II collagen, proteoglycans (90% aggrecan) and hyaluronic acid necessary for the assembly of cartilage matrix. Articular cartilage is frequently injured, often as a result of trauma and mechanical stress. Due to its limited capacity for repair, injured articular cartilage leads to chondral defects and subsequently develops into osteoarthritis. The incidence of chondral defects is very high in Australia, the United States and Europe. In a total of 31,000 arthroscopic surgical procedures reviewed, 63% were due to the chondral lesions with an average 2.7 lesions per knee (Curl et al, 1997, Arthroscopy, 13:456-460). Five percent of these occur in patients less than 40 years old. Articular cartilage defects can be graded accordmg to the Outerbridge classification (1961, J Bone Joint Surg 43:752-757) with modification. In grade I, the articular surface is swollen, soft and may be blistered. The lesion usually localizes at the superficial zone. Grade II is characterized by the presence of fissures and clefts measuring less than one centimeter in diameter and mainly occurs at both intermediate and deep zones. Grade III is characterized by the presence of deep fissures down to the calcified zone more than one centimeter in diameter. Loose flaps and joint debris also may be noticed. The defect in Grade IV occurs at the surface of subchondral bone. Clinically, there is little sign of spontaneous repair of hyaline cartilage for articular cartilage lesions. Various surgical interventions have been introduced to encourage the regeneration of cartilage defects (Browne et al. 2000, J Am Acad Orthop Surg, 8:180-189). However, almost all of them have failed to reconstruct the microarchitecture of articular cartilage. Procedures such as debridement, abrasion arthroplasty, subchondral bone drilling, osteochondral autograft and allograft may perhaps alleviate symptoms, but cannot restore the articular cartilage architecture.

Historically, there have been no effective surgical methods to restore hyaline articular cartilage and repair defects. Autologous chondrocyte implantation (ACI) or autologous chondrocyte transplantation therapy (ACTT) has been shown to be a promising method for restoring defects with hyaline cartilage. Since first reported by Brittberg et al. (1994, New Engl J Med, 331:889-895), studies on nine years of clinical follow-up indicated that ACTT has provided an excellent clinical outcome in the restoration of hyaline articular cartilage in majority (Peterson, et al, 2000, Alin Orthop, 374:212-234). The United States and European experience reported by the Cartilage Repair Registry of the Genzyme Tissue Repair Inc. (Cambridge MA), indicated that ACTT has achieved an 86% excellent outcome. The cumulative index rate of failure at two years was estimated at 5.8%.

The development of ACI represents the combined expertise of cell biologists and clinical orthopaedic surgeons. Chondrocytes for cultivation are taken from the cartilage of the non- weight bearing area of the proximal part of the condyle. A cartilage biopsy is subjected to enzymatic separation of the chondrocytes. Cells obtained are induced to differentiate into chondrocytes and propagated into 15 - 20 million cells within 2 - 3 weeks. These autologous chondrocytes are then placed (e.g., by being injected under a membrane or adhered to a membrane) into the defect site of articular surface.

Currently, there are two methods for covering the cartilage lesion. The original method, developed by Brittberg and Peterson, uses a periosteal flap whereas the advanced method, developed by a biotechnology company in Leverkusen, Germany, Verigen Transplantation Service International (VTSI) AG, uses a biodegradable collagen membrane prepared from type I and type III collagen for covering the defect surface. ACI has now been recommended for the treatment of clinically significant and symptomatic cartilage defects in the knee in the U.S., Europe, and Australia. Patients between 15 and 55 years old with a cartilage defect of one square centimeter to 10 square centimeters, and larger in area are suitable for the treatment. The Federal Drug Administration of the United States has approved this cell-based therapy as an alternative treatment for cartilage defects in the knee. In Australia, ACI is at stage IV of the assessment by the Medical Services Advisory Committee.

Although the concept of ACI has given rise to the new field of tissue engineering in orthopaedics, many questions remain to be answered. As ACI relies on the use of cultured cells and the biosynthetic profile of cultured chondrocytes has been shown to be altered during in vitro expansion, cultivation of chondrocytes for ACI has presented many technical and quality control challenges.

SUMMARY OF THE INVENTION The present invention includes methods for certifying chondrocytes for use in cartilage regeneration by assessing indicators of chondrocyte cell viability in a given chondrocyte cell culture. The present invention also includes production of a quality assurance certificate for a given chondrocyte cell culture.

In one embodiment of the present invention, a method for certifying chondrocyte cells for use in cartilage regeneration is provided. The method provides for collecting data indicating chondrocyte cell viability for use in cartilage regeneration and production of a certificate of chondrocyte cell viability including the data collected. A kit for quality assurance is also provided in the present invention. The kit includes instructions for collecting data indicating chondrocyte cell viability for use in cartilage regeneration and a certificate of chondrocyte cell viability including the data. In addition, a method for determining the likelihood of hyaline cartilage regeneration in a patient having a cartilage defect is provided in an embodiment of the present invention. The method comprises removing chondrocytes from said patient, culturing the chondrocytes, determining the presence of one or more differentiation factors and the percent apoptosis of the chondrocyte culture, and determining the likelihood of hyaline cartilage regeneration based on this information.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows a flow chart of one embodiment of a method for the characterization of cultured chondrocytes, according to the present invention.

Figure 2 depicts an MRI image of a patient having a partial cartilage defect.

Figure 3 is an image of a Western blot analysis of expression of TGF-beta-3 in chondrocyte cells. Figure 4 is an image depicting immunofluorescent detection of TGF- beta-3 in chondrocyte cells. Representative fluorescent cells are indicated by arrows.

Figure 5 is an image depicting apoptotic cells detected using TUNNEL assay. Representative apoptotic cells are indicated by arrows. Figure 6, comprising Figures 6A and 6B, shows apoptosis of chondrocyte cells as determined by flow cytometry in accordance with one embodiment of the present invention.

Figure 7 shows one embodiment of a certificate for chondrocytes according to the present invention. Figure 8 shows another embodiment of a certificate for chondrocytes.

Figure 9 shows yet another embodiment of a certificate for chondrocytes. DETAILED DESCRIPTION In one embodiment, the present invention is directed to alleviating one or more of the problems associated with the assessment of the cellular phenotype and predisposition to apoptosis of cultured chondrocytes, consistent with differentiation of articular hyaline cartilage.

It is further directed to a method of assessment of the cellular phenotype or viability of cultured chondrocytes, consistent with differentiation of articular hyaline cartilage. This is a requirement if delivery of ACI for restoration of hyaline cartilage is to be undertaken.

Surprisingly, it was determined that by using one or more of RT- PCR, Western blot, immuiiofluorescence/immunohistochemistry, flow cytometry and TUNNEL assay analyses, or other analytical tools, the viability of cultured chondrocytes for use in cartilage regeneration can be assessed, ultimately resulting in methods and kits for monitoring the viability of chondrocyte cells and providing a quality control of such cells.

Many factors control the differentiation of chondrocytes. For example, sex-determining factor Y, box 9 (SOX-9) is a transcriptional factor necessary for the induction of chondrogenesis. Cbfa-1 is another factor that is able to control differentiation of a chondrocyte toward an osteoblast. Growth factors which are capable of inducing chondrogenesis include Indian Hedgehog (Ihh), transforming growth factor-beta (TGF-beta 3), bone morphogenetic protein-2 (BMP-2), and parathyroid hormone-related peptide (PTHrP).

The present invention discloses methods for the characterization of cells and assessment of apoptosis of cells for use in cartilage regeneration. The disclosed methods are one aspect of a reliable and effective system for certifying the viability of cultured chondrocytes for implantation in patients with cartilage defects. In one aspect, the present invention provides a certificate of chondrocyte cells for each patient, and monitors the quality of the cultivation of chondrocytes in good manufacturing practice (GMP) approved laboratories.

In one aspect, the present invention relies on various methods, including reverse transcriptase polymerase chain reaction (RT-PCR), immunofluorescence/irnmunohistochemistry, and Western blot analysis, for the assessment of indicators associated with chondrogenesis. In addition, methods such as flow cytometry, fluorescence spectroscopy, and Tdt-Utp nick-end labeling (TUNNEL or TUNEL) assay, are used to assess the frequency of apoptosis in a given chondrocyte culture. Characterization of a chondrocyte culture and assessment of the rate of apoptosis have been significant steps toward the development of a quality assurance system for the cultivation of chondrocytes and to provide a kit that includes a certificate for chondrocytes.

In one embodiment of the present invention, a method for certifying chondrocyte cells for use in cartilage regeneration is disclosed. The method comprises collecting data indicating chondrocyte cell viability for use in cartilage regeneration and providing a certificate of chondrocyte cell viability including the collected data.

In this embodiment, the data collected which indicates chondrocyte cell viability includes the percent apoptosis. The percent apoptosis is one indicator of the capacity of the chondrocyte cell culture to regenerate hyaline cartilage. Knowing the percent apoptosis in a given chondrocyte cell culture is relevant because regeneration of cartilage depends, in part, on the number of chondrocyte cells implanted into a cartilage defect. Therefore, a cell culture that is highly apoptotic will not provide a solid support for cartilage regeneration. In fact, a chondrocyte culture which is greater than about 8% apoptotic indicates that a chondrocyte cell population will have difficulty in restoring hyaline cartilage as evidenced by magnetic resonance imaging (MRI). This is demonstrated in an experiment in which a 42-year old patient received autologous chondrocyte implantation with a chondrocyte cell population having 8.5% apoptotic chondrocytes and 91.5% normal chondrocytes. The patient experienced only partial regeneration of hyaline cartilage as evidenced by MRI, while patients having less than 8% apoptotic chondrocytes experienced full regeneration of hyaline cartilage. Figure 2 depicts partial cartilage regeneration in this patient. Thus, apoptosis of chondrocytes serves as an indicator for predicting cartilage regeneration in ACI. Cartilage regeneration, in addition to the degree of apoptosis, also relies on differentiation and proliferation of chondrocyte cells. Therefore, a method according to one embodiment of the present invention provides for assessment of expression of differentiation factors as an indication of chondrocyte cell viability for use in cartilage regeneration.

The indicators which are assessed in this method of the invention are one or more of those proteins related to the differentiation of chondrocyte cells. Preferably, one or more of the transcriptional factors, cytokines, including growth factors, or matrix proteins (collectively referred to herein as "differentiation factors") are assessed to predict the likelihood of satisfactory differentiation occurring. For example, one or more of the following differentiation factors selected from the group consisting of SOX-9, Cbfa-1, Ihh, TGF-beta-3, BMP-2, PTHrP, type I and type II collagen, aggrecan, and alkaline phosphatase, are assessed for expression in the chondrocyte cell culture. Assessment of these differentiation factors can be based on any one factor alone, or on a combination of factors. In one embodiment, the assessment is based on the expression of at least one of SOX-9 and aggrecan, for characterizing the chondrocyte cells. In another embodiment of the present invention, at least two differentiation factors are assessed for expression in the chondrocyte cell culture. In another embodiment of the present invention, at least three differentiation factors are assessed. In another embodiment, at least four differentiation factors are assessed. In another aspect of the present invention, five or more known chondrocyte differentiation factors are assessed.

Figure 1 is a flow chart demonstrating one embodiment of a method for the characterization of cultured chondrocytes, according to the present invention. In Figure 1, the method includes the steps of one embodiment for determining viability of cultured chondrocytes. This method includes step 100 culturing chondrocytes, step 110 assessing expression of at least one differentiation factor in a chondrocyte cell culture using, for example, RT-PCR, step 120 monitoring the apoptosis of a chondrocyte cell culture using, for example, Annexin- V flow cytometry, and step 130 filling in the assessment details on a certificate for chondrocytes. In the certificate produced in step 130, the presence or absence of chondrocyte differentiation factors and the apoptosis results are transcribed so that a clinician or scientist is able to quickly and easily assess the viability of the cultivated chondrocytes. Preferably, methods for assessing expression of differentiation factors include RT-PCR, Western blot, immunohistochemistry, or immunofluorescence. One or more of these methods are used in the methods and kits according to the present invention. An advantage of using immunohistochemistry or immunofluorescence methods to detect chondrocyte markers is that these methods can be used directly on chondrocytes of matrix- induced autologous chondrocyte implantation (MAO^®) as prepared by Verigen Transplantation Service International (VTSI; Leverkusen, Germany).

Protocols for each of these methods are provided in, for example, Sambrook et al. (1989, In: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York) and Ausubel et al. (1997, In: Current Protocols in Molecular Biology, Green & Wiley, New York), the entire content of which are incorporated herein by reference.

In addition to assessing expression of differentiation factors, the present invention also provides a method for assessing predisposition of a chondrocyte cell to apoptosis, thus providing medical practitioners, chondrocyte cultivation medical practitioners, and chondrocyte cultivation medical companies with a competent component of one embodiment of a testing system for ACI, according to the present invention. Thus, another embodiment of the present invention includes a kit for collecting data indicating the viability of a chondrocyte cell culture, a certificate, and instructions on collecting data and filling out the certificate.

Preferred methods for assessing apoptosis include, without limitation, fluorescence microscopy, flow cytometry, and TUNNEL assay. Annexin-V flow cytometry detection involves detection of cell- surface phosphatidylserine with Annexin V, which serves as a marker for apoptotic cells. The staining solution for use with Annexin V includes both the combination of Annexin- V-FLUOS andpropidium iodide and the combination of Annexin- V- Alexa 568 and BOBO-1. The cells are then analyzed in a flow cytometer or under a fluorescence microscope. Example 4 includes a flow chart (Table 4) of this procedure and the results obtained for flow cytometry analysis are shown in Figures 6A and 6B and are explained in Example 4. In Figures 6A and 6B, living cells are indicated in the lower left quadrant, apoptotic cells are indicated in the lower right quadrant, and necrotic cells are indicated in the upper right quandrant.

Cells may also be labeled with other membrane stains, such as a fluorescein- or phycoerythrin-labeled monoclonal antibody with either of the Annexin V stains.

In another embodiment of the present invention, the a method for determining the likelihood of full hyaline cartilage regeneration in a patient having a cartilage defect is described. The method includes removing chondrocyte cells from a patient, culturing the chondrocytes, and assessing the percent apoptosis in the chondrocyte cell culture. The methods described above for assessment of percent apoptosis are also useful in this embodiment of the present invention. In one embodiment, after determining the percent apoptosis, the resultant percent is compared to the 8 percent threshold. If the resultant percent apoptosis is higher than 8 percent, it is determined with a degree of assurance that the chondrocyte culture will not be capable of satisfactory hyaline cartilage regeneration. If the resultant percent apoptosis is lower than 8 percent, it is determined with a degree of assurance that the chondrocyte culture will be able to satisfactorily regenerate the hyaline cartilage within a cartilage defect.

The results obtained from assessing chondrocyte cell differentiation factors and analyzing percent apoptosis are transcribed onto the Certificate for Chondrocytes, an embodiment of which is shown in Figure 7. Alternatively, the characterization and apoptosis analyses can be reported separately. In another embodiment, a Certificate for Chondrocytes, shown in Figure 8, contains the results of the differentiation factors assessment only, and in another embodiment, a Certificate for Chondrocytes, shown in Figure 9, contains the results of the apoptosis analysis only. All Certificates for Chondrocytes include patient, hospital production manager, quality assurance officer, and/or surgeon identification information. In a preferred embodiment, the Certificate for Chondrocytes includes all such information. Certain aspects of the present invention will now be further described by way of reference to the following non-limiting examples for characterizing chondrocyte cells and determining predisposition of chondrocyte cells to apoptosis. It should be understood, however, that the examples following are illustrative only, and should not be taken in any way as a restriction on the generality of the invention described above.

EXAMPLE 1 : Characterization Of Chondrocytes Using RT-PCR RT-PCR was performed for several markers for chondrocyte differentiation, and PCR primers were developed using the nucleotide sequences of these markers, including collagen I (GenBank Accession No. XM 012651), collagen II (GenBank Accession No. L 10347), aggrecan (GenBank Accession No. XM 083921), SOX-9 (GenBank Accession No. XM 039094), BMP-2 (GenBank Accession No. NM 001200), TGF-beta-3 (GenBank Accession No. NM 003239), Cbfa-1. PTHrP (GenBank Accession Nos. M 57293, M 32740), alkaline phosphatase (GenBank Accession No. XM 001826), and Indian hedgehog. The primers and PCR conditions are shown in Table 1 and Table 2, respectively.

Total RNA was isolated from chondrocyte cultures using RNAzol solution according to the manufacturer's instructions (Ambion Inc., Austin, TX). For RT-PCR, single-stranded cDNA was prepared from 2 μg of total RNA using reverse transcriptase (Promega, Sydney Australia) with an oligo-dT primer. Two μl of each cDNA was subjected to 30 cycles of PCR using 1.0 unit of Taq polymerase (Promega, Sydney Australia) with 0.4 mMol/L of primers, 125 uMol/L of dNTP in lx PCR buffer, and water in a total volume of 25 μl (see Table 2). The amplification was performed in a DNA thermal cycler (Model 2400; Perkin-Elmer).

Specific primer sequences were selected from separate exons of the genes of interest, so as to avoid contamination of genomic DNA signal. Primers were designed using the software program at http://genzi.virus.kyoto-u.ac.jp/cgi- bin primer3.cgi and synthesized by Genset Oligos (Australia) at http://www.gensetoligos.com australia (see Table 1). As an internal control, the single stranded cDNA was PCR-amplified for 25 cycles using specific primers of a housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The PCR products were electrophoresed on 1.5% of agarose gel, stained with ethidium bromide. TABLE 1

TABLE 2

Protocol, IX reaction mix:

10X PCR buffer 2.5 μl dNTP (5 mM) 2.0μl sense primer (~15-25μM) 0.5μl (final concentration of

0.3-0.5 μM) antisense primer (-15-25 μM) 0.5μl ddH₂O 17.0μl

DNA Pol. 0.5μl cDNA 2.0μl

TOTAL 25.0μl

Cycle conditions used were:

EXAMPLE 2: Characterization of Chondrocytes Using Western Blot Analysis

Several markers for chondrocytes including type II collagen, aggrecan and S-100 protein, and other proteins, can be used to characterize cultured chondrocytes using Western blot analysis. Antibodies against such markers are commercially available, for example from Sigma (St. Louis, MO), Dako (AUSTRALIA) and R&D Systems (Minneapolis, MN).

The materials and methods for Western blot analysis of chondrocytes is now described in detail.

Cells were lysed by collecting about 10³ -10⁴ cultured chondrocytes and centrifuging them into a pellet. The supernatant was drawn off and the pellet was resuspended in 250 microliters of NET-gel Lysis Buffer (Quagen GmbH, Germany) and incubated 20 minutes on ice. Using a pipette, the cell debris and lysis buffer were transferred to a 1.5 milliliter Eppendorf™ tube and centrifuged at 12000g for 2 minutes at 4 degrees Celsius. The supernatant was removed to a new tube and an SDS-PAGE gel was run on the supernatant. The gel was transferred to a Hybond TM-C 0.45 μm nitrocellulose membrane (Amersham, Piscataway, NJ) using the Mini Trans-blot electrophoretic transfer cell (Bio-Rad, California, USA) at 30V (40mA) for overnight. The transfer is carried out in the presence of transfer buffer containing 7.57 grams of glycine, 369 grams of Tris and 400 milliliters of methanol in 2 liters of water (Sambrook et al. 1989, In: Molecular Cloning: A

Laboratory Manual, Cold Spring Harbor Laboratory Press, New York). Standard protocols for Western blot are available in, for example, Sambrook et al. (1989, In: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York) and Ausubel et al. (1997, In: Current Protocols in Molecular Biology, Green & Wiley, New York), which are hereby incorporated by reference Denaturation and Renaturation Step

Four solutions of guanidine-HCl (G-HC1) at concentrations of 6M, 3M, 1M, and 0.1M were prepared. Table 3 provides details for preparation of the G-HC1 solutions used in this step. When preparing the G-HC1 solutions, all ingredients should be prepared fresh. Milk powder was dissolved in water prior to adding it to the other ingredients to create a final concentration in the G-HC1 solution of 2% milk. The membrane was washed four times, thirty minutes per wash, once each with 6M G- HC1, 3M G-HC1, 1M G-HC1, and 0.1M G-HC1 at room temperature. The membrane was then washed with affinity chromotography (AC) buffer plus 2% milk powder solution overnight at 4 degrees Celsius.

AC buffer is prepared as follows: 50 mL glycerol (final 10% glycerol) 10 mL 5M NaCI (final lOOmM NaCI) 10 mL 1M Tris, pH 7.6 (final 20mM Tris) 1 mL 0.5M EDTA (final 0.5mM EDTA) 5 mL 10% Tween-20 (final 0.1% Tween-20) put on ice

Table 3: Guanidine-HCl Solutions for Denaturation/Renaturation Step

Washing and Blocking Step

The membrane was then washed two times with IX TBS-Tween for five minutes, followed by one wash with AC Buffer for five minutes. The membrane was then incubated for 1 hour at room temperature with a blocking solution prepared with 2% skim milk and IX TBS-Tween, followed by two five- minute washes with IX TBS-Tween. Probing for the Protein of Interest

The Probing Reaction Mixture (2% skim milk powder in 20 IX TBS-Tween with 50μL of Protein Probe and 20μL of 1M DTT) was added to the membrane and incubated for 2 hours at 4 degrees Celsius, followed by two washes with IX TBS-Tween for five minutes each wash at 4 degrees Celsius. The Protein Probe is the antibody against the protein of interest. In this case, the Protein Probe was antibody to TGF-beta-3. The membrane was then washed again with 10 mL of 2% skim milk in IX TBS-Tween for 15 minutes at 4 degrees Celsius, followed by a second wash with IX TBS-Tween for 20 minutes at 4 degrees Celsius. Addition of Primary Antibody

The membrane was washed two more times, five minutes each wash in IX TBS-Tween buffer using a rocking machine.

A 20 mL tube containing IX TBS-Tween and 1% skim milk (0.2 grams) was prepared and aliquotted into two 10 mL tubes, for primary and secondary antibody. One μL of anti-V5 antibody was pipetted into the primary antibody tube for' a final antibody dilution of 1/10000, and gently mixed. The primary antibody solution was poured onto the membrane and incubated on a rocking machine for 2 hours at room temperature. Alternatively, the antibody solution can be incubated overnight at 4 degrees Celsius. Addition of Secondary Antibody

After incubation, three washes with lxTBS-Tween, 5 minutes per wash were performed.

Five μL of secondary antibody (anti-mouse IgG-Fab) was pipetted into the secondary antibody solution for a final dilution of secondary antibody of 1/2000, and mixed gently. The secondary antibody solution was poured over the membrane and incubated for 45 minutes at room temperature on a rocking machine.

Addition of Detection Solution

After incubation with the secondary antibody solution, two washes were performed with lxTBS-Tween, for 5 minutes each wash on a rocking machine. Two more washes, each for 5 minutes were performed with IX TBS ONLY on the rocking machine.

The detection solution was prepared by mixing 2 mL of Lumigen Detection Solution A and 50 μL of Lumigen Detection Solution B (ECL plus, Sydney, Australia) and added to the membrane, making sure the membrane was evenly coated with the detection solution. The excess detection solution was shaken off, and the membrane was sealed in plastic wrap, making sure no wrinkles were present in the wrap. The membrane was placed on a piece of film in a film frame and exposed for about 30 minutes (exposure time will vary), then developed. Figure 3 demonstrates the results of Western Blot analysis using the method described above of detection of TGF-beta-3 in cultured chondrocytes. TGF-beta-3 is indicated.

EXAMPLE 3: Immunohistochemistry and Immunofluorescent Analysis

Similar to Western blot analysis, several markers for chondrocytes including type II collagen, aggrecan and S-100 protein can be used to characterize the cultured chondrocytes using immunohistochemistry and immunoflurorescence. These methods can be used directly on chondrocytes of MACI^® (matrix induced autologous chondrocyte implantation).

The materials and methods are now described. Chondrocytes on a MACI^® membrane are fixed with 5% paraformaldehyde solution and were subject to direct immunoflurorescence. Alternatively, the chondrocytes may be paraffin-embedded after fixation. The chondrocytes were then washed in 0.2M Tris-buffered saline (TBS), and blocked for endogenous peroxidase by incubation in 35% hydrogen peroxide (H₂O₂). The cells were then pre-incubated with 20% normal horse serum, and incubated with a first antibody. The cells were washed with TBS and incubated with a second antibody (which may be conjugated). A color reaction detection system such as 3 '3 '-diammobenzidine for detecting peroxidase conjugated with streptavidin is used to detect the chondrocyte markers.

Figure 4 demonstrates expression of TGF-beta-3 in chondrocytes cultured on a collagen membrane as detected by immunofluorescence using the above described method. In Figure 4, chondrocyte cells positive for TGF-beta-3 fluoresce, and such positive cells are indicated by an arrow. EXAMPLE 4: Apoptosis And Viability Of Chondrocytes Annexin- V method for assessment of apoptosis of chondrocytes

Annexin V is a phospholipid-binding protein with a high affinity for phosphatidylserine (PS). Detection of cell-surface PS with Annexin V or other membrane stain thus serves as a marker for apoptotic cells.

The materials and methods are now described.

Cultured chondrocytes were harvested, washed and pelleted. The chondrocytes were then resuspended in a staining solution containing Annexin-V- FLUOS and propidium iodide or Annexin- V-Alexa 568 and BOBO-1. Chondrocytes can also be labelled with other membrane stains, such as a fluorescein-, or phycoerythrin-labelled monoclonal antibody simultaneously with the Annexin stain. The cells were then analyzed in a flow cytometer or under a fluorescence microscope. A flow chart in Table 3, below, indicating the steps for testing chondrocyte apoptosis follows:

Table 3 Treat sample (10⁴-10⁶ cells) with apoptosis-inducing agent (1-24 h)

Wash treated cells with PBS and centrifuge (200 x g) (5 min, RT*)

Incubate cells in binding buffer containing Annexin- V-FLUOS and propidium iodide or Annexin- V-Alexa™ 568 and BOBO™-! (10-15 min, RT)

Add binding buffer to stained cells Analyze by fluorescence microscopy

Analyze by flow cytometry

*RT means room temperature

Results demonstrating flow cytometry analyses are shown in Figures 6A (case 1) and 6B (case 2). The following table 5 shows the summary data of flow cytometry analyses for Figures 6A and 6B. As can be seen, both patients have an apoptotic rate that is below the 8 percent threshold. Thus, both of these patients should have an excellent clinical outcome to fully regenerate cartilage defects.

Table 5

Living Cells Necrotic Cells Apoptotic Cells Cell Viability

Case 1 90% 4% 2% 90%

Case 2 92% 2% 6% 92%

TUNNEL method for assessment of apoptosis of chondrocytes The TUNEL reaction was prepared substantially in accordance with the instructions included with the Boehringer Mannheim TUNEL assay kit. Cells positive for apoptosis were stained dark purple by the end of the procedure and negative cells remained clear.

Figure 5 depicts results of a TUNEL assay, and positive, apoptotic cells are indicated with an arrow.

EXAMPLE 5: Certification of Chondrocytes

Data obtained from Examples 1 through 4 above is then used to produce a certificate of chondrocytes as exemplified in Figure 7, Figure 8, or Figure 9. The patient's name, date of birth, address, Patient ID number,

Hosptial, and surgeon in charge are indicated in the "Patient Information" section of the Certificate. After characterization of the patient's chondrocytes, either the "yes" or "no" box is checked next to the differentiation factor to indicate whether the factor is present in the chondrocyte culture. After the apoptotic studies of the patient's chondrocyte culture is completed, the percent apoptotic cells is indicated on the certificate, as well as the percent necrotic cells and the percent of cell viability. The Chief Scientist has the opportunity to write comments, and the certificate must be signed by the Production Manager and a Quality Assurance Officer.

Claims

CLAIMS What is claimed is:

1. A method for certifying chondrocyte cells for use in cartilage regeneration, said method comprising: a. collecting data indicating chondrocyte cell viability for use in cartilage regeneration; and b. providing a certificate of chondrocyte cell viability including the data.

2. The method of claim 1, wherein the data includes percent apoptosis.

3. The method of claim 2, wherein the percent apoptosis is determined using TUNNEL assay.

4. The method of claim 2, wherein the percent apoptosis is determined using flow cytometry.

5. The method of claim 2, wherein the percent apoptosis indicating viability is less than about 8 percent.

6. The method of claim 1, wherein the data includes an indicator of chondrocyte cell differentiation.

7. The method of claim 6, wherein the indicator includes a transcriptional factor.

8. The method of claim 7, wherein the transcriptional factor includes SOX-9.

9. The method of claim 7, wherein the transcriptional factor includes Cbfa-1.

10. The method of claim 6, wherein the indicator includes a cytokine.

11. The method of claim 10, wherein the cytokine includes a growth factor.

12. The method of claim 11, wherein the growth factor includes TGF-beta-3.

13. The method of claim 11, wherein the growth factor includes Indian hedgehog (Ihh).

14. The method of claim 11, wherein the growth factor includes bone morphogenetic protein-2 (BMP-2).

15. The method of claim 11, wherein the growth factor includes parathyroid hormone related peptide (PTHrP).

16. The method of claim 6, wherein the indicator includes alkaline phosphatase.

17. The method of claim 6, wherein the indicator includes a cartilage matrix protein.

18. The method of claim 17, wherein the matrix protein includes aggrecan.

19. The method of claim 17, wherein the matrix protein includes collagen type I.

20. The method of claim 17, wherein the matrix protein includes collagen type II.

21. The method of claim 6, wherein the indicator is assessed using Western blot analysis.

22. The method of claim 6, wherein the indicator is assessed using immunofluorescence.

23. The method of claim 6, wherein the indicator is assessed using RT-PCR.

24. A kit for quality assurance comprising: a. instructions for collecting data indicating chondrocyte cell viability for use in cartilage regeneration; and b. a certificate of chondrocyte cell viability including the data.

25. The method of claim 24, wherein the data includes a percent apoptosis.

26. The method of claim 25, wherein the percent apoptosis is determined using TUNNEL assay.

27. The method of claim 25, wherein the percent apoptosis is determined using flow cytometry.

28. The method of claim 25, wherein the percent apoptosis indicated viability is less than about 8 percent.

29. The method of claim 24, wherein the data includes an indicator of chondrocyte cell differentiation.

30. The method of claim 29, wherein the indicator includes a transcriptional factor.

31. The method of claim 30, wherein the transcriptional factor includes SOX-9.

32. The method of claim 30, wherein the transcriptional factor includes Cbfa-1.

33. The method of claim 29, wherein the indicator includes a cytokine.

34. The method of claim 33, wherein the cytokine includes a growth factor.

35. The method of claim 34, wherein the growth factor includes TGF-beta-3.

36. The method of claim 34, wherein the growth factor includes Indian hedgehog (Ihh).

37. The method of claim 34, wherein the growth factor includes bone morphogenetic protein-2 (BMP-2).

38. The method of claim 34, wherein the growth factor includes parathyroid hormone related peptide (PTHrP).

39. The method of claim 29, wherein the indicator includes alkaline phosphatase.

40. The method of claim 29, wherein the indicator includes a cartilage matrix protein.

41. The method of claim 40, wherein the matrix protein includes aggrecan.

42. The method of claim 40, wherein the matrix protein includes collagen type I.

43. The method of claim 40, wherein the matrix protein includes collagen type II.

44. The method of claim 29, wherein the indicator is assessed using Western blot analysis.

45. The method of claim 29, wherein the indicator is assessed using immunofluorescence.

46. The method of claim 29, wherein the indicator is assessed using RT-PCR.

47. A method for determining the likelihood of hyaline cartilage regeneration in a patient having a cartilage defect, said method comprising: a. removing chondrocytes from said patient; b. culturing said chondrocytes; c. determining the percent apoptosis of the chondrocyte culture in said step (b); and d. determining the presence of a differentiation factor; e. determining the likelihood of full hyaline cartilage regeneration by assessing the presence of the differentiation factor in said step (d) and comparing the percent apoptosis calculated in said step (c) with a known percentage, such that if the differentiation factor is present and the calculated percent apoptosis is greater than the known percentage, the likelihood of full hyaline cartilage regeneration is decreased.

48. The method of claim 47, wherein the percent apoptosis is assessed using flow cytometry.

49. The method of claim 47, wherein the percent apoptosis is assessed using TUNNEL assay.

50. The method of claim 47, wherein said chondrocytes are cultured on a membrane.