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IntroductionYounger people in the age group of 35–55 years with meniscal injuries, cartilage damage, and degenerative arthritis due to trauma are being diagnosed more commonly. Problems with young arthritic knees are dealt with very limited options. �e most frequently used reparative treatment for small symptomatic lesions of articular cartilage of the knee are marrow-stimulating techniques such as subchondral drilling, abrasion arthroplasty, and microfracturing [1, 2]. Bone marrow contains mesenchymal stem cells that have the potential to form new cartilage. However, �brocartilage typically forms, which is mechanically much less robust than articular
cartilage. Autologous cartilage implantation (ACI) and osteoarticular autogra� transfer are other more invasive modalities which are expensive, however, have not proven to be the “Gold Standard” for osteoarticular defects. Newer avenues are being researched for this ever-growing problem. �e reconstruction of cartilage defects using osteochondral allogra�s has been proven to be a suitable treatment for traumatic lesions only, but a�er longer periods, the viability of the allogra� decreases [3, 4]. Moreover, the immune response to the osteochondral allogra�s presents an unsolved problem [5]. Tissue engineering is an emerging interdisciplinary �eld that applies the principles of biology and engineering to the development of viable substitutes
¹Department of Orthopaedics , Institute of Medical Sciences, Banaras Hindu University,Varanasi-221005.²School of Biochemical Engineering, Indian Institute of Technology (Banaras Hindu University), Varanasi, India-221005.³Department of Biotechnology, Koneru Lakshmaiah Education Foundation, Vaddeswaram, Guntur, India-522502.
Address of Correspondence:Dr. Shivam Sinha, Assistant Professor Dept. of Orthopaedics, Institute of Medical Sciences, Banaras Hindu University,Varanasi- 221005Email: [email protected] Dr. Shivam Sinha Dr. Abhimanyu Madhual
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Background: Osteochondral lesions in young adults, if le� untreated invariably progresses to degenerative joint disorders. �e problem adds a burden on �nances and average productivity of the patients and countries’ economics. Among the known strategies for treatment, none has proven to be gold standard, and each one is having their own pros and cons. Osteochondral tissue engineering (OTE) offers a promising future where cultured chondrocytes can be grown on biocompatible biomaterial scaffolds and implanted on defects.Materials and Methods: We tested a novel three-dimensional scaffold using chitosan and poly-L-lactide (PLLA) in recommended proportion on animal experimental model. It was seeded with autologous chondrocytes harvested from tibial condyle of rabbits. 10 rabbits were implanted with this cell-seeded scaffold and were followed at 4, 8, and 12 weeks before being sacri�ced. Histological and gross examination was graded on a rank scale. It was compared between the implanted knee and contralateral control void. Values were examined statistically.Results: Outcome shows good quality of cartilage tissue, less �brotic growth, be�er amalgamation with the surrounding tissue, uptake at the implanted bed, and regular proliferative growth as early as 8 weeks. �e scaffold imparts structural stability to the chondrocytes. �e scaffold does not interfere with the normal healing process and was less in�ammatory by 12 weeks.Conclusion: OTE with bioscaffold using PLLA-chitosan and cultured chondrocytes has positive and promising outcomes in healing of cartilage defects.Keywords: Chondrocytes, Chitosan, Poly-l-lactide, Bioscaffold, Chondral lesion, Osteochondral tissue engineering.
Dr. Amit Rastogi Dr. Pradeep K Srivastava Dr. Sarada P Mallick
Autologous Cultured Chondrocytes Impregnated in �ree-dimensional Biodegradable Scaffold for Chondral Defects in Rabbits - An Experimental
Study
Sinha S et al www.jbjdonline.com
that restore, maintain, or improve the function of human tissues. Cartilage tissue engineering paradigm is based on the isolation of chondrocytes/chondrocyte precursors from a tissue biopsy, expanding the cell number in culture, seeding them onto three-dimensional (3D) scaffold, and incubating for a period of time before placing the construct inside a patient. Challenges include isolating, propagating chondrocytes, gaining relevant reproducible construct, morphology, size, and ensuring good durability of the construct in vivo before testing it on human subjects. Numerous scaffold materials such as agarose gel, poly-l-lactide (PLLA), gelatin, and chitosan have been evaluated by upcoming research for adherence to chondrocytes and imitating the anisotropy and mechanical properties of cartilage tissue with promising results. Arthritic or degenerative joint has a milieu of inhibitory growth factors which may increase the degeneration of scaffolds or inhibit the cells seeded on the bioscaffold. We hereby evaluated a novel 3D scaffold for adherence of autologous cultured chondrocyte in vitro, its ability to regenerate normal hyaline cartilage in articular defect created in rabbit’s knee, and its adverse reactions and biodegradability in vivo.
Materials and Methods Preparation of scaffoldDesirable scaffold characteristics include biocompatibility, porosity, optimal pore size, acceptable chemical, topographical, and mechanical properties. 3D scaffold was prepared using chitosan and PLLA. PLLA is a biodegradable and easily processable synthetic polymer that has good mechanical properties. Chitosan’s structure is similar to glycosaminoglycans, thus rendering it suitable for the cartilage tissue engineering. Chitosan, a semi-
crystalline polymer, composites mainly assist the a�achment, morphology, and proliferation of different lineage of the cells such as chondrocytes, hepatocytes, dermal �broblasts, and adrenal chromaffin cells. Chitosan solution with concentration of 2% (w/v) was �rst prepared by dissolution of the polymer in 2% glacial acetic acid solution. Similarly, 2% PLLA solution was prepared by dissolution in 2% (v/v) chloroform solution. Chitosan solution and PLLA solutions were mixed in the proportion of 70:30 on volume basis, as proposed by Mallick et al. [6]. �e solution was kept at −20°C for the duration of 24 h. �e whole mixture was kept in a lyophilizer at −50°C for 48 h to get a freeze-dried product. Freeze-drying technique enhances cross-linking of the two molecules (PLLA-chitosan) and makes it more stable in 3D state.
Procuring chondrocytes and preparation�e study was duly approved by Institutions Review Board for procuring the rabbits and performing experiments. 10 white New Zealand rabbits, adult males of average 1.5–2.0 kg, were chosen and le� for 2 weeks before starting the experiment. �e rabbits were kept in stainless steel cages (one animal per cage) at an ambient temperature of 22–27°C and relative humidity of 40–60% with 10 h:14 h light:dark cycle and maintained under pathogen-free conditions. �ey had free access to standard diet (pellet) and water ad libitum. �e principles of laboratory care, feeding, and sacri�ce of animal was followed as per ICMR guidelines on care of experimental animals. Operative sites of both the lower limbs of rabbits were shaved as they were operated simultaneously (Fig. 1). Rabbits were anesthetized using standard protocol (1 mg/kg ketamine hydrochloride and 1 mg/kg of midazolam) supplemented
Figure 3: Creating subchondral defect with 3 mm Steinmann's pin.Figure 2: Medial exposure of knee and medial tibial condyle.Figure 1: Prepared lower limb of rabbit for surgery.
Figure 4: Characteristics of chitosan-poly-L-LACTIDE scaffold. (a) Light microscopy reveals increased density of scaffold with 70:30 proportion, (b) scanning electron microscopy reveals improved porosity, (c) proliferation assay of cells on scaffolds a�er 3, 5, and 7 days of cell seeding.
Figure 5: Cell-seeded scaffold. Figure 6: Implantation of cell-seeded scaffold in subchondral defect.
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Sinha S et al www.jbjdonline.com
with 2% xylocaine with adrenaline (1:100,000) locally at the operative site. �e operative site, both the tibiae, was painted properly with standard aseptic precautions. Operative area was cleaned with 70% ethyl alcohol and povidone-iodine (10% aqueous solution). �e metaphysis of tibia was exposed by a 2 cm longitudinal incision along the medial aspect (Fig. 2). A�er retracting the so� tissues, a 2–3 mm unicortical defect was created in the medial condyle of proximal tibia using a Steinmann pin (Fig. 3). �e wound was irrigated with saline to remove clots and bone debris if any. Care was taken not to enter the subchondral bone and cause bleeding. �e chondrocytes were transported to laboratory in sterile medium containing phosphate buffer solution. �e wound was closed in layers and sterile dressing was applied. Postoperatively, rabbits were kept in separate cages and fed on standard diet. Post-operative antibiotics were administered. Dressing was changed on the 3rd post-operative day. �e tissues were washed with phosphate saline buffer. Cartilage tissue was minced using tissue homogenizer and treated with buffer. Sequential enzymatic digestion of this tissue was done using trypsin and collagenase, a�er dissolving them in Dulbecco’s modi�ed Eagle’s medium (DMEM) added with 1% antibiotic-antimycotic solution. At �rst, trypsin digestion was done in shaking �ask at humidi�ed 5% carbon dioxide environment. Following this, tissue was washed thrice with DMEM media and with the 10% fetal bovine serum (FBS). Collagenase treatment was done under similar conditions.
A�er complete digestion, the solution was �ltered through 70 µm sterile nylon mesh, and the �ltrate was subjected to centrifugation at 2500 rpm for 10 min. A�er centrifugation, supernatant was discarded and cell pellet was dissolved in DMEM media. Primary cell culture was done by maintaining the cells in tissue culture �ask under humidi�ed environment containing 5% CO2.
SubpassagingCon�uent monolayer of chondrocytes was obtained in 1–2 weeks of the chondrocytes culture. To obtain subpassages, cell monolayer was detached from the tissue culture �ask by treatment with the 0.25% of the trypsin solution. A�er trypsin treatment and monolayer detachment, trypsin is deactivated by FBS. �e cell suspension was centrifuged at 2500 rpm for 10 min. Cell pellet was obtained and dissolved in the fresh culture medium. Cell count and viability test were done using hemocytometer and trypan blue dye.
Cell scaffold seedingCells were seeded on sterile (70% ethanol treated) scaffold having a concentration of 1 × 10⁶ cells per ml by static method in a bioreactor. A bioreactor can be de�ned as a device that uses mechanical means to in�uence biological processes. In tissue engineering, bioreactors can be used to aid in the in vitro development of new tissue by providing biochemical and physical regulatory signals to cells and encouraging them to undergo differentiation and/or to produce extracellular matrix before in vivo implantation.
Standardization of ScaffoldProliferation assay, cell a�achment study, GAG (Glycosaminoglycans) quanti�cation, and porosity measurement showed optimal ratio of chitosan:PLLA was a ratio of 70:30, with electron microscopy con�rming the pore size of 38–172 µm and porosities in the range of
Figure 7: At 8 weeks, (a) control limb at 8 weeks showing non-healing defect, (b) partial uptake of scaffold in test sample at 8 weeks, (c) control sample grossly showing no healing at the defect site. Cartilaginous cap present at the junction, (d) test sample histopathology chondrocyte - regular growth, cartilaginous cap is thin. Scaffold - degraded proteinaceous material
Figure 8: At 12 weeks, (a) non-healing defect, (b) healed defect, (c) microscopic examination of control sample - no evidence of healing of any kind either �brotic or proliferative in control limb, (d) test limb histopathology - chondrocyte growth nodular proliferative growth at the junction with complete uptake at the gra� bed and coalition with the adjacent normal cartilaginous cap. Scaffold - visible.
Time of sacrificeTest group
(implanted)
Control group
(unimplanted)Total
4 weeks 2 2 4
8 weeks 3 3 6
12 weeks 3 3 6
Total 8 8 16
Table 1: Schedule of sacrifice of the rabbits
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73–93% (Fig. 4a-c) [6].
Scaffold implantation�e le� knee of the rabbit was taken as control and the right side as the test limb. �e le� side was not implanted with any scaffold while the right limb was implanted with cell-seeded scaffold. �e cultured autologous chondrocytes over the 3D biodegradable scaffolds were transferred into the defect by reopening the previously operated site (Figs. 5, 6). �e rabbits were followed up a�er a period 4, 8, and 12 weeks by gross and histological examination.
Gross and histological evaluationAt the end of 4, 8, and 12 weeks each, rabbits were sacri�ced with thiopental sodium (70 mg/kg) I/M. �e lower limbs were disarticulated from the knee joint and �xed in 10% buffered formalin for 3 days. Each specimen was decalci�ed and embedded in paraffin. Sections 4 µm thick were prepared and stained with hematoxylin and eosin. Microscopic examination was done under light microscope.
Statistical analysisFor the purpose of analysis of the data, the qualitative data obtained from the gross �nding, microscopy, and scaffold morphology at different periods of follow-up were assigned ranks/values, and the results were analyzed by non-parametric continuous Mann–Whitney U-test using SPSS, version 21.
Results A total number of 10 rabbits were operated, of which two died postoperatively due to infection. �erefore, observations of the present study are based on 16 limbs of eight rabbits, eight limbs of implanted group (test), and eight limbs of unimplanted group (control). Table 1 demonstrates the schedule of sacri�ce in test and control rabbits at 4, 8, and 12 weeks.Gross �ndings were assigned the values as follows:• Grade 0 = No healing of defect, defect visible to naked eye• Grade 1 = Partial or incompletely healed defect• Grade 2 = Completely healed defect.Histologic �ndings were graded as follows:• Grade 0 = Gap with no evidence of healing.
• Grade 1 = Incomplete healing of defect, marked by in�ammation or reparative process.• Grade 2 = Incomplete healing with haphazard/regular arrangement of chondrocytes with thin cartilaginous cap.• Grade 3 = Chondrocyte growth normal and regular or nodular with complete uptake at the bed as well as complete merging of new cartilage epithelium cells with the normal cartilaginous cap at the junction.Scaffold morphology graded as follows: • Grade 1 = Scaffold visible• Grade 2 = Visible but continuous with margins/appeared on bed• Grade 3 = Non-visualized.Scaffold histology was ranked as follows:• Grade 0 = Scaffold - present, not taken up. Signs of in�ammation and granulation tissue present but less �brosis.• Grade 1 = Partly degraded with proteinaceous material • Grade 2 = Not visible.Numerical �ndings at respective follow-up in the two groups are summarized in Table 2. Statistical analysis in between the test and control group reveals signi�cance only at 8 weeks (P = 0.025) and 12 weeks (P = 0.034) within 95% of con�dence interval.
Discussion Despite all the recent advances in cartilage tissue engineering, the forage for a superlative method to augment cartilage regeneration continues. High-density cultures promote chondrogenic differentiation of totipotent mesenchymal cells. �e close spatial relation of surrounding chondrocytes provides a feasible environment for cell-cell and cell-matrix interactions maintaining differentiated chondrocytes. To deliver the cultured chondrocytes to the created defect, we devised a new technique in which cultured chondrocytes were impregnated on PLLA and chitosan biodegradable scaffolds which enhanced the cell proliferation by various biomechanical stimuli. �e method has demonstrated high potential to produce normal cartilage in vivo. Since articular cartilage has limited regeneration potential, its inadequate repair will lead to long-standing pain and morbidity in the patient. Of all contemporary cartilage preserving techniques, ACI has gained popularity in the treatment of focal articular cartilage defects in the early 1990s and received widespread use clinically. Following in vitro cultivation, the suspension of chondrocytes is injected into a pocket created by suturing the periosteal �ap to the surrounding cartilage. �e lack of a perfect seal of the defect leads to leakage of the cell suspension with treatment failure rates being signi�cant in procedures not done properly. Grande et al. examined the
Sinha S et al www.jbjdonline.com
Rabbit No.Follow-up
weeks
Gross HistopathologyGross
specimen
Scaffold
morphology
Scaffold
histologyHistopathology
A4 4 0 0 1 1 1 1
B4 4 0 1 1 1 0 1
C8 8 0 1 1 2 1 2
D8 8 0 0 1 2 1 2
E8 8 0 1 1 2 1 2
F12 12 1 1 2 3 2 3
G12 12 0 0 2 3 1 3
H12 12 0 1 2 3 2 3
Control Test Test
Table 2: Gross and Histological grading of specimens
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effect of autologous chondrocytes grown in vitro on the healing rate of chondral defects that did not penetrate the subchondral bone using nuclear tracer chondrocytes. In defects that had received transplants, a signi�cant amount of cartilage regenerated (82%) determined using qualitative and quantitative light microscopy. Autoradiography con�rmed the presence of autologous labeled cells in the newly formed regenerated matrix [7]. Briitberg et al. used a similar rabbit model to transplant autologously harvested and in vitro cultured chondrocytes into patellar chondral lesions (size <3 mm) that had been made previously and were extending down to calci�ed zone, without using any bioscaffold. Chondrocytes transplantation signi�cantly increased the amount of newly formed repaired tissue [8]. Transplantation of chondrocytes or chondrogenic cells alone was shown to be successful in rabbit models, but the healing rate was limited due to loss of viability in the transplanted cells and due to the difficulty of �xing chondrocytes in the defect [3, 7, 9]. Merely, transplanting cultured chondrocyte was not adequate to obtain normal function. Drawback of ACI includes subsidence of osteochondral lesion, inadequate hyaline cartilage formation, and persistence of deeper lesion. �erefore, urging researchers to develop scaffolds strong enough to sustain load-bearing conditions. Strategies for transplantation of chondrocytes or chondrogenic cells seemed to play a speci�c role since these cells are able to regenerate a functional and biomechanically intact extracellular matrix and to preserve its functional stability for a longer period. One of such strategies is to use a scaffold material. Bri�berg et al. used autologous chondrocyte, cultured chondrocyte and chondrocyte-seeded carbon �ber pad scaffold in rabbit knees, and compared healing at 12 weeks and 52 weeks with controls in which periosteal �ap was done over chondral lesions in patella. �ey summarized, histological grading was improved in all knees with chondrocyte treatment, yet carbon scaffold was incompletely bonded to adjacent cartilage at 1 year. Our results show statistically signi�cant integration of scaffold by 8 weeks, and within 12 weeks, there was adequate cartilage formation (Figs. 7, 8) [10]. Early scaffolds by Kawamura et al. used collagen gel - allogenic chondrocytes in a similar experimental study. A�er 2 weeks of culture in vitro, they found improved elasticity and stiffness. At 6 months, histology demonstrated good repair in gel - allogenic chondrocytes implanted knee as early as 1 day continued up to 6 months [11]. Similar studies by Rahfoth et al. on agarose gel and Plonczak on polysulfonic membrane found promising results with cultured chondrocytes [12, 13]. In contrast to the present study, Boopalan et al. in their study on rabbits using allogenic chondrocytes concluded that
chondral defects treated with allogenic chondrocyte transplantation resulted in improved hyaline characteristics of regenerated cartilage. To avoid immunological reactions and ethical issues, usage of autologous chondrocytes was emphasized instead of allogenic ones in the present study [14]. Choice of biomaterials for fabrication of integrated scaffolds for tissue engineering is challenging [15, 16]. Each layer of the scaffold must be engineered to exhibit tissue-speci�c biophysical conditions and environment and to support the regeneration of two distinct cell types: Chondral tissue and subchondral osteogenic tissue separately. To enhance bone regeneration, ceramic materials, such as hydroxyapatite (HAp) or β-tricalcium phosphate (TCP), are o�en incorporated into the portion of the bilayered scaffold that would generate new bone. For instance, Sherwood et al. used 3D printing to produce PLGA/PLA scaffolds for cartilage region and PLGA/TCP scaffolds for bone regeneration and integrated them into a bilayered scaffold using salt-leaching technique [17]. Holland et al. used a two-stage cross-linking procedure for oligo(poly(ethylene glycol) fumarate (OPF) and OPF/gelatin microparticles [18]. Collagen and collagen/PLGA bilayered scaffold were integrated by combination of solvent casting and salt-leaching and freeze-drying techniques [19]. Fabrication of chitosan/HAp-chitosan was done by sintering/freeze-drying techniques and particle aggregation methodology in recent researches [20, 21]. Chitosan was preferred in our study as recent research has shown that it increases mineralization during osteoblast differentiation of human bone marrow-derived mesenchymal stem cells by upregulating genes associated with collagen type 1 alpha 1, integrin-binding sialoprotein, osteopontin, osteonectin, and osteocalcin, signi�cantly [22]. �e present research work is aimed at transplantation of autologous chondrocytes seeded on biodegradable templates of PLLA and chitosan on focal subchondral defects. By implanting scaffolds instead of injecting suspensions, our technique allows for expansion of cells before transplantation, and the scaffolds being rigid and smooth apart from being biodegradable are prefabricated to �t the defect increasing their retention and washout which was a disadvantage in injecting suspensions. �e eventual outcome showed good quality of cartilage tissue, less �brotic growth, be�er amalgamation with the surrounding tissue, uptake at the bed, and regular proliferative growth, by 8 and 12 weeks. �e scaffold imparts structural stability to the chondrocytes. It does not interfere with the normal healing process since it is biodegradable. Our studies have certain limitations. First, a smaller sample size as sacri�cing animals on a larger scale had ethical issues. Second, a long-term follow-up histopathology up to an year (52 weeks)
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would have made results comparable to other studies and the quality of hyaline cartilage analysis. Finally, the results cannot be extrapolated to human cartilage regeneration unless trial is done.
Conclusion�e use of our novel-3 D PLLA-chitosan scaffold with
autologous chondrocytes in tibial condylar defects in rabbits has shown exciting early results, by 12 weeks, in terms of production of hyaline cartilage, adequate preservation of biology by the biomaterials, and absence of tissue reactions. �is seems to promise its use for human trials as a breakthrough in osteochondral tissue engineering (OTE).
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References
Con�ict of Interest: Nil. Source of Support: None
How to Cite this ArticleSinha S, Madhual A, Rastogi A, Srivastava P K, Mallick S P. Autologous Cultured Chondroc y tes Impregnated in �ree-dimensional Biodegradable Scaffold for Chondral Defects in Rabbits - An Experimental Study. Journal of Bone and Joint Diseases Sep - Dec 2018;33(3):29-34.
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