Intra-individual comparison of human nasal chondrocytes and debrided knee chondrocytes: Relevance for engineering autologous cartilage grafts

Abstract

OBJECTIVE:

Implantation of autologous chondrocytes for cartilage repair requires harvesting of undamaged cartilage, implying an additional joint arthroscopy surgery and further damage to the articular surface. As alternative possible cell sources, in this study we assessed the proliferation and chondrogenic capacity of debrided Knee Chondrocytes (dKC) and Nasal Chondrocytes (NC) collected from the same patients.

METHODS:

Matched NC and dKC pairs from 13 patients enrolled in two clinical studies (NCT01605201 and NCT026739059) were expanded in monolayer and then chondro-differentiated in 3D collagenous scaffolds in medium with or without Transforming Growth Factor beta 1 (TGFβ1). Cell proliferation and amount of cartilage matrix production by these two cell types were assessed.

RESULTS:

dKC exhibited an inferior proliferation rate than NC, and a lower capacity to chondro-differentiate. Resulting dKC-grafts contained lower amounts of cartilage specific matrix components glycosaminoglycans and type II collagen. The cartilage forming capacity of dKC did not significantly correlate with specific clinical parameters and was only partially improved by medium supplemention with TGFβ1.

CONCLUSIONS:

dKC exhibit a reproducibly poor capacity to engineer cartilage grafts. Our in vitro data suggest that NC would be a better suitable cell source for the generation of autologous cartilage grafts.

Keywords

Nasal chondrocytes cartilage repair tissue engineering chondrogenic differentiation articular cartilage

1 Introduction

Articular cartilage is a tissue with limited intrinsic capacity to repair, so that cartilage lesions often fail to heal on their own and may be associated with pain, loss of function and long-term complications such as osteoarthritis [1]. An established method to treat articular cartilage lesions consists on the implantation of ex vivo cultured autologous knee chondrocytes. For such approach, usually a biopsy in a healthy area of the already damaged joint is collected to derive the therapeutic cells [2]. This procedure, however, represents an additional injury to the cartilage surface and has been reported to be detrimental to the surrounding healthy articular cartilage [3] and to be associated with increased pain perception in some patients [4].

Nasal cartilage would be an interesting alternative source of chondrocytes, considering that it can be harvested under local anesthesia and by a procedure that is less invasive than removing tissue from specific areas of the joint. Moreover, in vitro expanded nasal chondrocytes (NC) have been shown to generate cartilaginous tissues with higher and more reproducible quality than those generated with articular chondrocytes (AC) and to be capable of adapting to the biological and physical environment of a load-bearing joint [5 –8]. The implantation of tissue engineered cartilage grafts based on autologous NC has been recently demonstrated to be safe and feasible for knee cartilage repair [9]. Evidences of the efficacy of this treatment are being collected in an ongoing multicenter phase II clinical trial (clinicaltrials.gov NCT026739059) under the lead of the University Hospital of Basel within the EU-funded project “BIO-CHIP” (http://biochip-h2020.eu).

A possible problem associated with this aforementioned strategy is represented by the required involvement of rhinoplasty surgeons for the collection of the nasal cartilage biopsy, that might increase the logistic and complexity of the therapeutic approach.

Alternatively, debrided tissue (which is usually discarded) could be collected by orthopedic surgeons at the edges of the lesioned cartilage, thus avoiding the iatrogenic damage of the harvesting procedure, and used to retrieve articular chondrocytes. Indeed, few reports provided proof of principles that viable chondrocytes, competent to proliferate and chondro-differentiate can be isolated from damaged cartilage areas [10 –12]. However, the quality of the chondrocytes that can be isolated from such damaged cartilage area might quite substantially vary according to the extent/chronicity of the lesion and the status of the patient’s joint. Indeed it might be possible that chondrocytes resident at the site of chronic cartilage lesions in some patients have acquired alterations compromising their therapeutic utility.

In the context of the aforementioned past and ongoing clinical trials, we collected from the same patients specimens from the nasal cartilage septum in a first intervention (to acquire the NC for the manufacturing of the cartilage graft) and from the damaged knee cartilage in a second intervention (to refresh the cartilage lesion before the placement of the cartilage graft). We aimed this study at comparing the capacity of NC and debrided knee chondrocytes (dKC) isolated from the corresponding tissue specimens to engineer cartilage grafts. Additionally, we sought to identify possible correlations between the cartilage forming capacities of the dKC and specific clinical parameters (i.e.: number of previous interventions, onset of symptoms prior to surgery) and subjective view reported by the patient related to the knee status (i.e., Knee injury and Osteoarthritis Outcome Score (KOOS) on pain, other symptoms related to the knee, function in daily living, function in sport and recreation, and knee-related quality of life).

2 Materials and methods

2.1 Collection of samples

The samples described below (n = 13) were collected from patients enrolled in a concluded Phase I clinical trial (clinicaltrials.gov NCT01605201) or an ongoing Phase II clinical trial (clinicaltrials.gov NCT02673905). The clinically relevant information related to these patients are reported in Table 1. Briefly, patients were aged between 26 and 52 years (mean age of 39±10 years), 11 out of 13 patients underwent 1-2 previous operations (median 1 operation, mainly addressing meniscal tear repairs or debridement), and 6 out of 13 patients had cartilage lesions in 2 locations in average (i.e.: femoral condyle and trochlear). Size of the cartilage injuries were between 2.7 and 7.0 cm² (4.3±1.4 cm²). Most patients had cartilage lesions due to sport injuries. Clinically, patients presented symptoms such as pain, swelling of the joint or blocking sensation, which were graded in the KOOS (Knee Osteoarthritis Outcome Score) scale, as a value out of the maximum of 100 points (meaning no perceived problems). Preoperative mean scores for KOOS subscale “Symptoms” were 59.1±22.4 points, “Pain” 60.9±23.0 points, “Activity of daily living” 65.5±24.6 points. Most severe impairment in knee function was related to the categories “Sports” (33.8±26.2 points) and “Quality of life” (32.2±18.0 points). Patients had no previous history of infectious or systemic inflammatory diseases of the joints (exclusion criteria for the clinical studies).

Table 1
Clinical data

Patient No Gender Age (year) BMI Onset of symptomes (months) Previous OP No. Previous sport injuries Concomitant pathology Total size of lesion (cm²) Localization ICRS grade injuries Preoperative KOOS scores

Symptoms Pain ADL Sport QOL

1 M 46 30.1 49 1 Y varus>5° 4.5 FC, TF 3 42.9 44.4 57.4 5 12.5

2 M 29 28.4 30 1 Y none 3.0 FC 3 53.6 47.2 67.7 25 25.0

3 M 40 25.0 14 2 Y meniscus tear 7.0 FC, TF 4 53.6 55.6 66.2 35 37.5

4 F 50 21.0 14 1 Y none 6.5 FC, TF 3 17.9 19.4 20.6 5 6.3

5 M 40 29.0 1 2* Y meniscus tear 4.0 TF 3 39.3 47.2 35.3 30 25.0

6 M 29 26.0 15 2 N loose bodies 5.5 FC, TF 3 53.6 47.2 61.7 15 25.0

7 M 26 23.0 84 2 N meniscus tear 3.8 FC 4 78.6 91.7 97.1 95 25.0

8 F 52 21.6 5 1 N none 2.0 FC 4 92.9 86.1 85.3 35 56.3

9 M 52 24.7 9 0 Y none 3.4 FC, TF 4 85.7 100 100 60 68.8

10 M 36 25.4 10 0 Y none 4.0 FC, TF 4 92.9 80.6 76.5 55 56.3

11 M 41 26.9 10 1 Y meniscus tear 4.5 TF 4 39.3 55.6 63.2 40 25.0

12 M 25 27.7 14 1 Y meniscus tear 2.7 FC 4 46.4 38.9 27.9 0 31.3

13 M 40 28.4 192 1 N meniscus tear 5.0 FC 4 71.4 77.8 92.7 35 25.0

Patient No	Gender	Age (year)	BMI	Onset of symptomes (months)	Previous OP No.	Previous sport injuries	Concomitant pathology	Total size of lesion (cm²)	Localization	ICRS grade injuries	Preoperative KOOS scores
1	M	46	30.1	49	1	Y	varus>5°	4.5	FC, TF	3	42.9	44.4	57.4	5	12.5
2	M	29	28.4	30	1	Y	none	3.0	FC	3	53.6	47.2	67.7	25	25.0
3	M	40	25.0	14	2	Y	meniscus tear	7.0	FC, TF	4	53.6	55.6	66.2	35	37.5
4	F	50	21.0	14	1	Y	none	6.5	FC, TF	3	17.9	19.4	20.6	5	6.3
5	M	40	29.0	1	2*	Y	meniscus tear	4.0	TF	3	39.3	47.2	35.3	30	25.0
6	M	29	26.0	15	2	N	loose bodies	5.5	FC, TF	3	53.6	47.2	61.7	15	25.0
7	M	26	23.0	84	2	N	meniscus tear	3.8	FC	4	78.6	91.7	97.1	95	25.0
8	F	52	21.6	5	1	N	none	2.0	FC	4	92.9	86.1	85.3	35	56.3
9	M	52	24.7	9	0	Y	none	3.4	FC, TF	4	85.7	100	100	60	68.8
10	M	36	25.4	10	0	Y	none	4.0	FC, TF	4	92.9	80.6	76.5	55	56.3
11	M	41	26.9	10	1	Y	meniscus tear	4.5	TF	4	39.3	55.6	63.2	40	25.0
12	M	25	27.7	14	1	Y	meniscus tear	2.7	FC	4	46.4	38.9	27.9	0	31.3
13	M	40	28.4	192	1	N	meniscus tear	5.0	FC	4	71.4	77.8	92.7	35	25.0

Symptom onset is referring to the age of the cartilage pathology. Total size was measured according to intraoperative debridement. KOOS “Symptoms” subscale are assessing swelling of the joint, stiffness or blocking sensation.^*Patient 5 has been operated on the same knee but in another compartment (lateral meniscus), while the cartilage lesion on the trochlea and new meniscal lesion in the medial compartment was due to a recent and adequate football injury. Abbreviations: BMI: Body Mass Index, M: Male, F: Female, Y: Yes, N: No, FC: Femoral Condyle, TF: Trochlea Femoris, ICRS: International Cartilage Repair Society, KOOS: Knee Injury and Osteoarthritis Outcome Score, ADL: Activities of Daily Living, QOL: Quality Of Life.

A cartilage biopsy (6 mm diameter) was harvested from the nasal septum of 13 patients as previously described [9]. Knee articular cartilage samples were collected from the same patients as described below.

In the patients under general- or regional anesthesia, an arthrotomy of the knee was performed to expose the whole cartilage lesion(s) as well as adjacent cartilage tissue. The cartilage lesions were debrided according to the standard technique. Briefly, the area of the damaged cartilage was demarcated with a cartilage punch in order to include all damaged tissue in order to create a “shoulder” of healthy cartilage for later cartilage graft fixation with sutures. The demarcated cartilage defect mainly contained damaged cartilage tissue but could also contain some healthy tissue depending of the geometry of cartilage defect and the cartilage punch. Afterwards, the demarcated cartilage tissue – including thus both damaged and healthy tissue – was removed down to the calcified layer with a curette spoon, and this debrided tissue was kept apart as the sample. The collected scratching materials included only cartilaginous tissue. Samples were kept in a sterile tube containing a sterile saline solution and were transported immediately to the laboratory for further processing.

2.2 Chondrocyte isolation, expansion, and culture in 3-D scaffolds

2.2.1 Nasal chondrocytes (NC)

The digestion of nasal cartilage tissues and the culture of nasal chondrocytes for the manufacturing of the patient grafts were performed in the Good Manufacturing Practice (GMP) facility at the University Hospital Basel according to standard operating procedures under a quality management system, as described by Mumme et al. [9]. In brief, cartilage tissues were chopped in small pieces that were treated for 22 hours with NB6 GMP grade collagenase (5 U/ml final, Serva). The resulting cells were then cultured in monolayer for two passages in Expansion Medium (EM: Dulbecco’s modified Eagle’s medium [DMEM; Gibco] containing 4.5 mg/ml D-glucose and 0.1 mM non essential amino acids and supplemented with 1 mM sodium pyruvate [Gibco], 10 mM HEPES buffer [Gibco], 100 units/mL penicillin, 100 mg/ml streptomycin, Glutamax [Invitrogen] and supplemented with 5% autologous serum, 5 ng/ml Fibroblast Growth Factor 2 [FGF-2, GMP grade, R&D Systems Minneapolis, MN, USA] and 1 ng/ml Transforming Growth Factor β1 [TGF-β1; Promocell]). Cells were incubated for 2 weeks at 37°C and 5% CO₂ with media changes twice/week. Passage 2 (P2) cells were then seeded on collagen type I/III membranes (Chondro-Gide^®; Geistlich Pharma AG, Wolhusen, Switzerland) at a density of 4.17 million cells per cm². The resulting constructs were cultured for two weeks in Chondrogenic Medium (ChM: DMEM [Gibco] containing 4.5 mg/ml D-glucose and 0.1 mM non essential amino acids and supplemented with 1 mM sodium pyruvate [Gibco], 10 mM HEPES buffer [Gibco], 100 units/ml penicillin, 100 mg/ml streptomycin, Glutamax [Invitrogen] and supplemented with 5% autologous serum, 10 μg/ml insulin [Novo Nordisk, Bagsvaerd, Denmark], and 0.1 mM ascorbic acid 2-phosphate [Sigma-Aldrich]) at 37°C and 5% CO₂ with media changes twice/week for two weeks. P2 cells in excess and parts of the grafts that were not used for implantation in the cartilage defects were analysed in the laboratory. In case the entire graft was used for implantation in the patients, left-over NC at P2 were expanded for one additional passage, and the P3 cells cultured in Chondro-Gide membrane under the aforementioned conditions, with the only difference that Foetal Bovine Serum (FBS) instead of autologous serum was used for the EM and ChM.

2.2.2 Debrided knee chondrocytes (dKC)

Cartilage fragments from the same 13 patients were digested as described for the nasal cartilage samples and the isolated cells expanded in a biosafety level 2 cell culture laboratory for 2-3 passages in EM containing 5% FBS. Expanded cells were then cultured in Chondro-Gide membranes as described above, with ChM containing 5% FBS. dKC from three donors were also cultured in ChM supplemented with 10 ng/mL TGF-β1.

2.3 Analytical methods

2.3.1 Histology

Constructs were fixed in 4% paraformaldehyde, dehydrated, and embedded in paraffin. Safranin-O/fast green (SafO/FG) staining with hematoxylin (J.T. Baker) nuclear counterstaining was performed to analyze cartilage tissue formation. Semi quantitative assessment of the chondrogenesis was based on a simplified version of the Bern Score [13] (named Modified Bern Scored, MBS), that is currently implemented in the phase II clinical trial to assess the quality of the graft. For the MBS, two categories were considered for the scoring, each with equal weight, with a possible minimum collective score of 0 and a maximum of 6. These scoring categories are: “intensity of cell staining (0: no stain; 1 weak staining; 2 moderately even staining: 2; even dark stain: 3) and “cell morphology” (0: condensed/necrotic/pycnotic bodies; 1: spindle/fibrous; 2: mixed spindle/fibrous with rounded chondrogenic morphology; 3: majority rounded/chondrogenic). In order to score the quality of the engineered cartilage grafts, histological sections of the tissues were assessed in the following way: each histological section was divided in 3 parts and in each part three predefined areas were graded through a microscope (Widefield Microscope Olympus IX83) using a 10×objective. The MBS was then derived averaging the 9 obtained values. For the release of the engineered cartilage graft in the aforementioned clinical study, a MBS superior to 3.0 isconsidered.

Sections from engineered cartilage tissues were also processed for immunohistochemistry using an antibody against type II collagen (Abcam 34715, 1:1000) as previously described [14].

2.3.2 Quantification of glycosaminoglycans and DNA

Engineered grafts were digested with 1 mg/ml protease K in 50 mM Tris with 1 mM EDTA, 1 mM iodoacetamide and 10 mg/ml pepstatin-A for 16 hours at 56°C. For glycosaminoglycan (GAG) quantification, the method of Barbosa et al. (2003) [15] was used. Briefly, diluted or undiluted digested constructs (depending on SafO intensity) were incubated with 1 ml of dimethylmethylene blue assay (DMMB) solution (16 mg/l dimethylmethylene blue, 6 mM sodium formate, 200 mM GuHCL, pH 3.0) on a shaker at room temperature for 30 minutes. Precipitated DMMB-glycosaminoglycans (GAGs) complexes were centrifuged and supernatants were discarded. Complexes were dissolved in decomplexion solution (4 M GuHCL, 50 mM Na-Acetate, 10% Propan-1-ol, pH 6.8) at 60°C, absorption was measured at 656 nm and GAG concentrations were calculated using a standard curve prepared with purified bovine chondroitin sulfate. DNA content was measured by using the CyQuant Cell Proliferation Assay Kit (Molecular Probes Inc., Eugene, OR) according to the instructions of the manufacturer.

2.3.3 Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR)

Total RNA was extracted from expanded cells and from engineered cartilage grafts. cDNA synthesis and qRTPCR (7300, Applied Biosystems) were performed as previously described [16] to quantify expression levels of type I collagen (Col I, Hs00164004), type II collagen (Col II, Hs00264051), aggrecan (Agg, Hs00153936_m1), and Versican (Ver, Hs00171642_m1) (all from Applied Biosystems). For each sample, the Ct value of each target sequence was subtracted from the Ct value of the reference gene (human GAPDH, Hs02758991, Applied Biosystems) to derive the ΔCt. We used the ratios of mRNA levels of collagen type II to I (Col II/Col I) and of aggrecan to versican (Agg/Ver) as ‘differentiation indexes’ [17].

2.3.4 Statistical analyses

Statistical evaluation was performed using SPSS software version 22 software (SPSS, Sigma Stat). Values are presented as mean±SD (standard deviation of the mean). Differences between cell sources were assessed by two-tailed Student’s t-test. P values less than 0.05 were considered statistically significant. Correlations between MBS and patients’ parameters were assessed using two-tailed Pearson’s tests. P values less than 0.05 were considered to indicate statistically significantcorrelations.

3 Results

3.1 Proliferation rate and de-differentiation stage acquired by dKC and NC

Proliferation rate was estimated after the 2nd (P2) or 3rd (P3) replate. As reported in Fig. 1A, P2 dKC and NC proliferated at comparable rates (0.71±0.03 and 0.79±0.09 doublings/day, respectively). At P3, instead, dKC proliferated at statistically significant lower rates (0.66±0.06 and 0.91±0.08 doublings/day, respectively). RT-PCR analyses were also performed in expanded cells to assess extent of cell de-differentiation. Results reported in Fig. 1B showed that the collagen type II to I (Col II/Col I) and of aggrecan to versican (Agg/Ver) ratios at the mRNA level were similar for both cell sources (2.4^*10^–6 ±1.5^*10^–6 and 3.1^*10^–1 ^± 2.1^*10^–1 respectively for dKC and 1.7^*10^–5 ^± 1.0^*10^–5 and 1.5^*10^–1 ^± 0.7^*10^–1 respectively for NC).

Fig.1

Proliferation capacity and de-differentiation of debrided knee chondrocytes (dKC) and nasal chondrocytes (NC) during monolayer culture. (A) Proliferation rate of dKC and NC at passage 2 and 3. (B) Real-time reverse transcriptase– polymerase chain reaction analysis of post-expanded chondrocytes, the ratios of mRNA levels of collagen type II to I (Col II/Col I) and of aggrecan to versican (Agg/Ver) are reported. E = exponential in base 10. Values are the mean±standard deviation of cells from 13 different donors. ^* = P < 0.05 versus the corresponding dKC group.

3.2 Re-differentiation of expanded dKC and NC in Chondro-Gide membrane

The re-differentiation potential of expanded dKC and NC was evaluated by culturing the cells into Chondro-Gide membranes in a chondrogenic medium not containing TGF-β1 (since the absence of growth factors for the manufacturing of Advanced Therapy Medicinal Products, ATMPs, would be preferred). Both dKC and NC were capable to re-differentiate as demonstrated by an increase in Col II/Col I and Agg/Ver mRNA ratios (Fig. 2A vs Fig. 1B). However, NC acquired a significant superior extent of re-differentiation, as demonstrated by the higher values of both ratios in these cells as compared to dKC (102.2- and 16.1-fold higher Col II/Col I and Agg/Ver mRNA, respectively).

Fig.2

Chondrogenic differentiation of post expanded debrided knee chondrocytes (dKC) and nasal chondrocytes (NC). Post-expanded chondrocytes were cultured for two weeks onto collagen I/III scaffolds (Chondro-Gide^®) in chondrogenic medium not containing Transforming Growth Factor beta-1 (TGFβ-1). (A) Real-time reverse transcriptase– polymerase chain reaction analysis of constructs, the ratios of mRNA levels of collagen type II to I (Col II/Col I) and of aggrecan to versican (Agg/Ver) are reported. E = exponential in base 10. (B) Glycosaminonoglycans (GAG), DNA and GAG/DNA contents of genererated constucts. Values are the mean±standard deviation of constructs generated with cells from 13 different donors. ^* = P < 0.05 versus the corresponding dKC group.

Tissues generated by dKC and NC were also characterized biochemically. As reported in Fig. 2B, while grafts exhibited similar DNA/wet weight (0.23±0.03 and 0.19±0.02 μg/mg), the amounts of accumulated GAG/wet weight and GAG/DNA were statistically significant lower in grafts generated by dKC (0.60±0.06 vs 5.72±1.51 μg/mg and 2.72±0.63 vs 26.21±6.42 μg/μg, respectively). Finally, tissues were analysed histologically using the MBS scoring system. Scores related to the intensity of staining and morphology were inferior in the dKC-based grafts as compared to the NC-based grafts. The total MBS scores (max: 6) averaged 1.8±0.6 and 5.6±0.7, respectively (Fig. 3A).

Fig.3

Histological characterization of the constructs generated with debrided knee chondrocytes (dKC) and nasal chondrocytes (NC). Constructs were generated as described in the Legend of Figure 2. (A) Grading of the constructs performed using the Modified Bern Score (see Material and Methods section for the description), results related to the categories “intensity of cell staining” and “cell morphology” and the total are reported. Values are the mean±standard deviation of cells from 13 different donors. P < 0.05 versus the corresponding dKC group. (B - C) (immuno)histological characterization of constructs. Safranin O (B) and type II collagen (C) staining of worst and best quality constructs generated with dKC and NC. The insets in (B) are low-magnification images of the constructs.

Safranin-O staining of “worst” and “best” constructs (Fig. 3B) demonstrated that GAG positive areas were scattered and faintly stained in the “best” (but absent in the “worst”) dKC-based grafts, whereas rather strong and homogenous deposition of GAG could be observed even in the ”worst” NC-based graft. Immunohistochemical characterization of corresponding grafts showed that type II collagen was undetectable even in the “best” dKC-based graft but strongly expressed in all NC-based grafts (Fig. 3C).

Due to the poor cartilage forming capacities of all analyzed dKC, we could not find any significant correlation between chondrogenic capacity of the dKC (GAG/DNA ratios) and clinical parameters/scores. Nevertheless, we observed trends toward lower GAG content of constructs generated with dKC from patients that had previous sport injuries (30.2% reduction in GAG/DNA) or from patients that experienced more pain, other symptoms related to the knee and reduction of function in sport (i.e.: 31.6%, 22.5% and 22.5% decrease in GAG/DNA of patients with lower KOOS pain, symptom and sport, respectively) (Fig. 4).

Fig.4

Correlation between GAG/DNA contents of constructs and clinical patients’ parameters and scores. Values are the mean±standard deviation of constructs (N = 4 – 9). N.s.: not statistically significant. See legend to Table 1 for the description of the other abbreviations.

In order to assess whether medium supplementation with TGFβ1 could enhance the cartilage forming capacity of the dKC, debrided chondrocytes from three patients (#1, 2 and 4) were chondrogenically cultured in the absence or presence of this growth factor. Biochemical results demonstrated that while DNA contents remained unchanged in constructs cultured without or with TGFβ1, GAG contents were significantly higher in the latter constructs (average increase in GAG/wet weight of 3.0-fold, p < 0.05). However, GAG amounts of TGFβ1-cultured dKC remained 28.6-fold lower (p < 0.05) than those of corresponding tissues generated with NC in absence of TGFβ1. Grading of the SafO/FC stained tissue demonstrated a slight (1.3-fold), although not statistically significant, increase in MBS of engineered tissues generated with TGFβ1 (Table 2). In addition, even in presence of TGFβ1 the dKC produced no visually detectable amounts of Col II (Table 2).

Table 2

Biochemical and histological data

Patient #	Experimental groups	DNA/wet weight (μg/mg)	GAG/wet weight (μg/mg)	MBS (range: 0–6)	Collagen II staining intensity^&
1	dKC (-TGFβ1)	0.19±0.04	0.47±0.05°	2.2±0.3°	–
	dKC (+TGFβ1)	0.20±0.02	1.36±0.10*°	2.8±0.3°	–
	NC (-TGFβ1)	0.25±0.03	9.69±0.72*	4.5±0.5*	++
2	dKC (-TGFβ1)	0.19±0.03	0.42±0.15°	2.2±0.6°	–
	dKC (+TGFβ1)	0.22±0.02	1.49±0.22^*°	2.7±0.3°	–
	NC (-TGFβ1)	0.17±0.03	11.43±0.26^*	5.8±0.3^*	++
4	dKC (-TGFβ1)	0.19±0.03	0.54±0.08°	2.0±0.8°	–
	dKC (+TGFβ1)	0.18±0.02	1.37±0.18^*°	2.5±0.3°	–
	NC (-TGFβ1)	0.19±0.01	20.35±0.80^*	6.0±0.0^*	++

Biochemical and histological results of constructs generated with dKC cultured in the presence or absence of TGFβ1 and of those generated with the corresponding NC (as control). ^* = p < 0.05 from dKC (-TGFβ1), °= p < 0.05 from NC; -: no detectable, +: weak/moderate, ++: intense.

4 Discussion

In this study, using cartilage samples collected from the same patients, we showed that dKC as compared to NC exhibit (i) a reduced proliferation rate, (ii) a similar tendency to de-differentiate during the monolayer expansion and (iii) a significantly inferior re-differentiation capacity once cultured in 3D with a chondrogenic medium not containing TGFβ1, thus producing tissues with limited or undetectable cartilage matrix components (GAG and type II collagen). The supplementation of TGFβ1 to the chondrogenic medium resulted in a limited improvement of the quality of the constructs with regard to GAG, but still contained no detectable type II collagen.

At passage 2, proliferation rate of dKC was comparable to that measured in a previous study using cadaveric chondrocytes isolated from macroscopic unaffected knee cartilage (18). However, proliferation capacity of dKC remained constant or slightly decreased at passage 3 (in contrast to NC); further characterization is required to understand whether this behavior is a consequence of an early acquisition of senescence traits by chondrocytes derived from a biochemically and mechanically altered joint compartment. This would also explain the limited capacity of dKC to chondro-differentiate as observed in our study, considering that senescence in cartilage has been shown to impair chondrocyte anabolism [19]. In particular, constructs generated by dKC did not contain the main chondrogenic marker type II collagen. Immunoreactivity for type II collagen in tissues generated by debrided articular chondrocytes was instead reported by others [10, 11]. This discrepancy could be explained by the different procedure used to harvest the debrided articular chondrocytes. Indeed, this surgical step is not standardized. As a consequence, the debrided specimens collected from different surgeons during the refreshing of the cartilage lesions might drastically differ in the relative amounts of cartilage tissue of different types (i.e.: hyaline vs calcified) and pathological states (“damaged” vs “healthy”, derived respectively from the lesioned or from the adjacent unaffected areas).

In this study, autologous NC-based grafts were manufactured using autologous serum, while dKC-based grafts were generated using FBS. The reduced cartilage forming capacity of dKC- vs NC-based grafts is not likely due to this difference considering that a previous study demonstrated similar performance of chondrocytes cultured with FBS or human serum [20].

We showed that a minor improvement of cartilage maturation was achieved using TGFβ1 during the culture of dKC. However, GAG amounts remained relatively low and type II collagen virtually absent. To check the quality of the engineered cartilage graft we used the MBS, a histological scoring system currently used in the ongoing Phase II clinical trial as release criteria. In particular, engineered cartilage grafts are released for implantation if MBS > 3.0. According to this criterion, none of the dKC-based graft could be considered adequate for clinical use. The chondrogenic capacity of dKC could be improved by using different bioactive molecules [21, 22] or culturing the cells under hypoxic conditions [23 –25]. However, such conditions, especially the use of bioactive molecules, would be difficult to implement for the GMP-compliant manufacturing of the grafts. Nevertheless, we have to consider that, even if in principle a more abundant cartilage matrix within the grafts would guarantee a superior protection of the chondrocytes from the inflammatory and biomechanical insults, yet no clinical data are available to understand whether the implantation of more developed cartilage grafts (as compared to less mature ones) would promote a superior cartilage repair in patients. An ongoing international phase II clinical trial (BIO-CHIP) under the lead of the University Hospital Basel is formally testing the hypothesis that maturation of the implanted cartilage graft will improve the clinical efficacy.

In our relatively small cohort of patients for this study, no statistically significant correlation between the chondrogenic capacity of the dKC constructs and epidemiologic parameters or subjective scores of the patients was observed. However, we observed a trend towards an inferior chondrogenic capacity of dKC from patients with lower KOOS score subcategories, reinforcing the importance of taking in consideration the subjective evaluation of the patient as index of the joint status [26].

Importantly, even if chondrocytes from damaged cartilage could be considered an adequate cell source for the repair of focal cartilage, these cells are not suitable for the treatment of OA lesions. This is due to the difficulty to harvest reasonable amounts of cartilage from the affected OA knee to get sufficient numbers of cells and to the fact that the performance of the chondrocytes could be even lower if harvested from OA joints, though controversial reports are available in literature [27 –29]. Instead, NC can be easily harvested from a healthy cartilage compartment (the nose) of OA patients and used to generate high quality cartilage grafts that might be effective in the management of the OA lesions (unpublished data).

In conclusion, the present study demonstrates that articular chondrocytes isolated from debrided knee cartilage exhibit a reproducibly poor capacity to chondro-differentiate, even when cultured in the presence of the strong chondrogenic factor TGFβ1, indicating that these cells might not represent an adequate source for the engineering of implantable cartilage graft for cartilage repair, as, instead, is the case for nasal chondrocytes. Further characterizations are necessary to investigate which are the molecular mechanisms responsible for such impairment. Additionally, investigations would be required to assess whether the utilization of a laser technology [30] to refresh cartilage defects (instead of the standard methods relying on surgical skills and instrumentation) would allow to acquire articular chondrocytes with the capacity to generate grafts of superior properties.

Footnotes

Acknowledgments

We thank S. Saftic (Geistlich) for providing Chondro-Gide collagen scaffolds and A. Wixmerten for critical editing of the manuscript. This project was partially funded by the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 681103, BIO-CHIP (to IM) and by the Personenförderung program at the Department of Surgery of the University Hospital Basel (to GL).

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