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A cell‐free approach with a supporting biomaterial in the form of dispersed microspheres induces hyaline cartilage formation in a rabbit knee model

Authors:
Javier Zurriaga at IMED Hospitales
Javier Zurriaga
Maria laura Lastra at National University of La Plata
Maria laura Lastra
Carmen M. Antolinos‐Turpin
Carmen M. Antolinos‐Turpin
Carmen M. Antolinos‐Turpin
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Rosa Morales Román at Cetenma
Rosa Morales Román
  • Cetenma
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Abstract and Figures

The objective of this study was to test a regenerative medicine strategy for the regeneration of articular cartilage. This approach combines microfracture of the subchondral bone with the implant at the site of the cartilage defect of a supporting biomaterial in the form of microspheres aimed at creating an adequate biomechanical environment for the differentiation of the mesenchymal stem cells that migrate from the bone marrow. The possible inflammatory response to these biomaterials was previously studied by means of the culture of RAW264.7 macrophages. The microspheres were implanted in a 3 mm‐diameter defect in the trochlea of the femoral condyle of New Zealand rabbits, covering them with a poly(l‐lactic acid) (PLLA) membrane manufactured by electrospinning. Experimental groups included a group where exclusively PLLA microspheres were implanted, another group where a mixture of 50/50 microspheres of PLLA (hydrophobic and rigid) and others of chitosan (a hydrogel) were used, and a third group used as a control where no material was used and only the membrane was covering the defect. The histological characteristics of the regenerated tissue have been evaluated 3 months after the operation. We found that during the regeneration process the microspheres, and the membrane covering them, are displaced by the neoformed tissue in the regeneration space toward the subchondral bone region, leaving room for the formation of a tissue with the characteristics of hyaline cartilage.
(a) FESEM picture of PLLA microspheres produced by an oil/water emulsion (dimension bar 20 μm, the diameter of these microspheres was 40 ± 20 μm, mean ± SD); (b) binocular loupe pictures of CHT microspheres produced by neutralization of the CHT acidic solution swollen in water (dimension bar 200 μm, the diameter of these microspheres was 205 ± 5 μm, mean ± SD). CHT, chitosan; FESEM, field emission scanning electron microscopy; PLLA, poly(l‐lactic acid)
(a) FESEM picture of PLLA microspheres produced by an oil/water emulsion (dimension bar 20 μm, the diameter of these microspheres was 40 ± 20 μm, mean ± SD); (b) binocular loupe pictures of CHT microspheres produced by neutralization of the CHT acidic solution swollen in water (dimension bar 200 μm, the diameter of these microspheres was 205 ± 5 μm, mean ± SD). CHT, chitosan; FESEM, field emission scanning electron microscopy; PLLA, poly(l‐lactic acid)
… 
Stress–strain compression test of the wet microspheres confined in a plunger cylinder device (see the text). (•) Chitosan, CHT; () poly(l‐lactic acid), PLLA; and () PLLA+CHT
Stress–strain compression test of the wet microspheres confined in a plunger cylinder device (see the text). (•) Chitosan, CHT; () poly(l‐lactic acid), PLLA; and () PLLA+CHT
… 
MTT assay results expressed as % basal, taking as basal the cell viability under control conditions at 24 hr. RAW 264.7 macrophages cultured for 24 and 48 hr with chitosan, CHT; poly(l‐lactic acid), PLLA; and CHT + PLLA microspheres. Data represent the mean ± SEM
MTT assay results expressed as % basal, taking as basal the cell viability under control conditions at 24 hr. RAW 264.7 macrophages cultured for 24 and 48 hr with chitosan, CHT; poly(l‐lactic acid), PLLA; and CHT + PLLA microspheres. Data represent the mean ± SEM
… 
Cytotoxicity assay. Production of interleukin‐1β (IL‐1β) (a) and NO (b) by RAW 264.7 macrophages upon incubation with different microspheres. Data represent the mean ± SEM. *p < .05 versus control. NO, nitric oxide
Cytotoxicity assay. Production of interleukin‐1β (IL‐1β) (a) and NO (b) by RAW 264.7 macrophages upon incubation with different microspheres. Data represent the mean ± SEM. *p < .05 versus control. NO, nitric oxide
… 
Microscopic views of the experimental areas for groups (a) PLLA (H&E), (b) PLLA sample with a higher magnification showing the remaining microspheres and membranes located in subchondral bone (H&E), (c) PLLA + chitosan, PLLA + CHT (H&E), (d) series M (H&E), (e) sample of group PLLA + CHT stained with toluidine blue in order to evaluate metachromasia, and (f) image viewed under polarized light microscopy of a sample of the PLLA group. White arrows show the limit of the practiced cartilage defect, and yellow arrows and green arrows indicate some of the PLLA microspheres and the membrane remaining after 3‐month implantation. Individual scores in the ICRS II scale for each category and group are summed up in Table 2. CHT, chitosan; H&E, hematoxylin–eosin; ICRS, International Cartilage Repair Society; PLLA, poly(l‐lactic acid)
+2
Microscopic views of the experimental areas for groups (a) PLLA (H&E), (b) PLLA sample with a higher magnification showing the remaining microspheres and membranes located in subchondral bone (H&E), (c) PLLA + chitosan, PLLA + CHT (H&E), (d) series M (H&E), (e) sample of group PLLA + CHT stained with toluidine blue in order to evaluate metachromasia, and (f) image viewed under polarized light microscopy of a sample of the PLLA group. White arrows show the limit of the practiced cartilage defect, and yellow arrows and green arrows indicate some of the PLLA microspheres and the membrane remaining after 3‐month implantation. Individual scores in the ICRS II scale for each category and group are summed up in Table 2. CHT, chitosan; H&E, hematoxylin–eosin; ICRS, International Cartilage Repair Society; PLLA, poly(l‐lactic acid)
… 
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ORIGINAL RESEARCH REPORT
A cell-free approach with a supporting biomaterial in the form
of dispersed microspheres induces hyaline cartilage formation
in a rabbit knee model
Javier Zurriaga Carda
1,2
| Maria L. Lastra
3
| Carmen M. Antolinos-Turpin
4
|
Rosa M. Morales-Román
4
| María Sancho-Tello
1,5
| Sofía Perea-Ruiz
4
|
Lara Milián
1,5
| Juan M. Fernández
3
| Ana M. Cortizo
3
| Carmen Carda
1,5,6
|
Gloria Gallego-Ferrer
4,6
| José L. Gómez Ribelles
4,6
1
Departamento de Patología, Facultad de
Medicina y Odontología, Universitat de
València, Valencia, Spain
2
IMED (Innovación MÉDica), Hospital IMED,
Valencia, Spain
3
Laboratorio de Investigaciones en Osteopatías
y Metabolismo Mineral (LIOMM), Facultad de
Ciencias Exactas, Universidad Nacional de La
Plata 47 y 115 (1900), La Plata, Argentina
4
Center for Biomaterials and Tissue
Engineering (CBIT), Universitat Politècnica de
València, Valencia, Spain
5
INCLIVA Biomedical Research Institute,
Valencia, Spain
6
Biomedical Research Networking Center on
Bioengineering, Biomaterials and
Nanomedicine (CIBER-BBN), Valencia, Spain
Correspondence
José L. G. Ribelles, Center for Biomaterials and
Tissue Engineering (CBIT), Universitat
Politècnica de València, Valencia, Camino de
Vera s/n 46022 Valencia, Spain.
Email: jlgomez@ter.upv.es
Funding information
Comisión de Investigaciones Científicas de la
Provincia de Buenos Aires (CICPBA),
Universidad Nacional de La Plata, Grant/Award
Number: 11/X643; Agencia Estatal de
Investigación/Fondo Europeo de Desarrollo
Regional de la Unión Europea, Grant/Award
Number: MAT2016-76039-C4-1 2-R; Spanish
Ministry of Economy and Competitiveness
(MINECO)
Abstract
The objective of this study was to test a regenerative medicine strategy for the
regeneration of articular cartilage. This approach combines microfracture of the sub-
chondral bone with the implant at the site of the cartilage defect of a supporting bio-
material in the form of microspheres aimed at creating an adequate biomechanical
environment for the differentiation of the mesenchymal stem cells that migrate from
the bone marrow. The possible inflammatory response to these biomaterials was pre-
viously studied by means of the culture of RAW264.7 macrophages. The micro-
spheres were implanted in a 3 mm-diameter defect in the trochlea of the femoral
condyle of New Zealand rabbits, covering them with a poly(L-lactic acid) (PLLA) mem-
brane manufactured by electrospinning. Experimental groups included a group where
exclusively PLLA microspheres were implanted, another group where a mixture of
50/50 microspheres of PLLA (hydrophobic and rigid) and others of chitosan
(a hydrogel) were used, and a third group used as a control where no material was
used and only the membrane was covering the defect. The histological characteristics
of the regenerated tissue have been evaluated 3 months after the operation. We
found that during the regeneration process the microspheres, and the membrane
covering them, are displaced by the neoformed tissue in the regeneration space
toward the subchondral bone region, leaving room for the formation of a tissue with
the characteristics of hyaline cartilage.
KEYWORDS
articular cartilage regeneration, cartilage engineering, chitosan, microspheres, polylactide,
rabbit knee model
1|INTRODUCTION
Cartilage regeneration is a problem yet to be solved in clinical practice
because it is a tissue with a low cell density and without
vascularization. Osteochondral injuries often result in articular carti-
lage damage and premature osteoarthritis. This is already a huge prob-
lem, as it has been described to affect over 10.2% of adult population
(Carmona, Ballina, & Gabriel, 2001), and its effect is noted in a fivefold
Received: 25 February 2019 Revised: 29 July 2019 Accepted: 17 August 2019
DOI: 10.1002/jbm.b.34490
1428 © 2019 Wiley Periodicals, Inc. J Biomed Mater Res. 2020;108B:1428–1438.wileyonlinelibrary.com/journal/jbmb
... When the cells are implanted either encapsulated in gels or in the pores of a scaffold, the space available does not allow them to generate this organization and therefore it is necessary that the degradation of the implanted biomaterial leaves the necessary space for the formation of new tissue. Tissue organization is greatly enhanced when the implanted biomaterial is progressively displaced at the site of regeneration, as has been observed in rabbit knee models in which both scaffolds and dispersed material were implanted [3,16,17]. ...
... In a previous study we showed that the combination of subchondral bone stimulation and the implantation of a microgel formed by a combination of rigid microspheres of polylactic acid (PLA) and others made of chitosan, induces the formation of well-organized hyaline cartilage because the formation of the neotissue progressively displaces the microspheres towards the subchondral bone, leaving room for the macroscopic organization of the tissue produced by the MSCs that migrated from the bone marrow [17,18]. Chitosan has been used in different chondrogenesis studies but, on the one hand, it does not present recognizable sequences for the cells and is little or not adherent to them and, on the other hand, for clinical translation it has the disadvantage of the variability of its bioresorption in the organism since it is only degraded by enzymes such as lysozyme [19][20][21]. ...
... Chitosan has been used in different chondrogenesis studies but, on the one hand, it does not present recognizable sequences for the cells and is little or not adherent to them and, on the other hand, for clinical translation it has the disadvantage of the variability of its bioresorption in the organism since it is only degraded by enzymes such as lysozyme [19][20][21]. Moreover, certain anomalous effects were attributed to the presence of chitosan, such as the formation of disordered cartilaginous lumps on the articular surface, or the formation of cysts in the bone [17]. In this study we want to demonstrate the effectiveness of the strategy when a microgel formed by a mixture of PLA microspheres and others produced by the coagulation of platelet-rich plasma (PRP) obtained from the animal's circulating blood, is implanted at the site of regeneration. ...
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... On the other hand, it allows mesenchymal cells to easily migrate from the subchondral bone if the implantation of the support material is combined with an injury to the subchondral bone with techniques such as microfracture. This strategy allowed the formation of a tissue with all the characteristics of hyaline cartilage in a rabbit knee model [23]. ...
Mesenchymal Stem Cells Cultured in a 3D Microgel Environment Containing Platelet-Rich Plasma Significantly Modify Their Chondrogenesis-Related miRNA Expression
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The aim of this work is to study the effect of platelet factors on the differentiation of mesenchymal stem cells (MSCs) to hyaline cartilage chondrocytes in a three-dimensional environment. MSCs were cultured in a microgel environment with a chondrogenic medium. The microgel consisted of microspheres that combine gelatin and platelet-rich plasma (PRP). The gelatin/PRP microdroplets were produced by emulsion. The gelatin containing the microdroplets was enzymatically gelled, retaining PRP and, just before seeding the cells, platelets were activated by adding calcium chloride so that platelet growth factors were released into the culture media but not before. Platelet activation was analyzed before activation to rule out the possibility that the gelatin cross-linking process itself activated the platelets. The gene expression of characteristic chondrogenic markers and miRNA expression were analyzed in cells cultured in a differentiation medium and significant differences were found between gelation/PRP microgels and those containing only pure gelatin. In summary, the gelatin microspheres effectively encapsulated platelets that secreted and released factors that significantly contributed to cellular chondrogenic differentiation. At the same time, the microgel constituted a 3D medium that provided the cells with adherent surfaces and the possibility of three-dimensional cell–cell contact.
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... The cells are seeded into the space between the microspheres, whose surface can be functionalised to provide cell adhesion sequences [14][15][16], while the microspheres themselves can support the release of growth factors during culture [17,18]. The same strategy can be used for in vivo tissue regeneration [19]. ...
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Alginate hydrogels can be used to develop a three-dimensional environment in which various cell types can be grown. Cross-linking the alginate chains using reversible ionic bonds opens up great possibilities for the encapsulation and subsequent release of cells or drugs. However, alginate also has a drawback in that its structure is not very stable in a culture medium with cellular activity. This work explored the stability of alginate microspheres functionalised by grafting specific biomolecules onto their surface to form microgels in which biomimetic microspheres surrounded the cells in the culture, reproducing the natural microenvironment. A study was made of the stability of the microgel in different typical culture media and the formation of polyelectrolyte multilayers containing polylysine and heparin. Multiple myeloma cell proliferation in the culture was tested in a bioreactor under gentle agitation.
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... Recently, bioactive drugs loaded CS microspheres (CMs) advanced their osteogenic potential in tissue regeneration and controlled drug delivery applications [15]. Additionally, CMs has been employed to synthesize CMs/polymer scaffold for the treatment of bone defects [16]. Li et al. reported that adiponectin-loaded CMs embedded in PLGA/β-TCP scaffold increased bone formation and mineralization [17]. ...
Synergistic anti-inflammatory and osteogenic n-HA/resveratrol/chitosan composite microspheres for osteoporotic bone regeneration
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The development of functional materials for osteoporosis is ultimately required for bone remodeling. However, grafts were accompanied by increasing pro-inflammatory cytokines that impaired bone formation. In this work, nano-hydroxyapatite (n-HA)/resveratrol (Res)/chitosan (CS) composite microspheres were designed to create a beneficial microenvironment and help improve the osteogenesis by local sustained release of Res. Study of in vitro release confirmed the feasibility of n-HA/Res/CS microspheres for controlled Res release. Notably, microspheres had anti-inflammatory activity evidenced by the decreased expression of pro-inflammatory cytokines TNF-α, IL-1β and iNOS in RAW264.7 cells in a dose dependent manner. Further, enhanced adhesion and proliferation of BMSCs seeded onto microspheres demonstrated that composite microspheres were conducive to cell growth. The ability to enhance osteo-differentiation was supported by up-regulation of Runx2, ALP, Col-1 and OCN, and substantial mineralization in osteogenic medium. When implanted into bone defects in the osteoporotic rat femoral condyles, enhanced entochondrostosis and bone regeneration suggested that the n-HA/Res/CS composite microspheres were more favorable for impaired fracture healing. The results indicated that optimized n-HA/Res/CS composite microspheres could serve as promising multifunctional fillers for osteoporotic bone defect/fracture treatment.
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... In recent years, many studies have demonstrated the effects of COX-2 inhibitors on bone, however, the effects of COX-2 inhibitors (such as Etoricoxib) on subchondral bone in OA are still unclear, especially the role of COX-2 inhibitors on its microstructure [11,[22][23][24]. The microscopic biomechanical environment of knee subchondral bone is very important for the repair/degeneration of subchondral bone cells [25]. ...
Etoricoxib decreases subchondral bone mass and attenuates biomechanical properties at the early stage of osteoarthritis in a mouse model
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Etoricoxib, a selective Cyclooxygenase-2 (COX-2) inhibitor, is commonly used in osteoarthritis (OA) for pain relief, however, little is known about the effects on subchondral bone. In the current study, OA was induced via destabilization of the medial meniscus (DMM) in C57BL/6 mice. Two days after surgery, mice were treated with different concentrations of Etoricoxib. Four weeks after treatment, micro computed tomography (Micro-CT) analysis, histological analysis, atomic force microscopy (AFM) analysis, and scanning electron microscopy (SEM) were performed to evaluate OA progression. We demonstrated that Etoricoxib inhibited osteophyte formation in the subchondral bone. However, it also reduced the bone volume fraction (BV/TV), lowered trabecular thickness (Tb.Th), and more microfractures and pores were observed in the subchondral bone. Moreover, Etoricoxib reduced the elastic modulus of subchondral bone. Exposure to Etoricoxib further increased the empty/total osteocyte ratio of the subchondral bone. Etoricoxib did not show significant improvement in articular cartilage destruction and synovial inflammation in early OA. Together, our observations suggested that although Etoricoxib can relieve OA-induced pain and inhibit osteophyte formation in the subchondral bone, it can also change the microstructures and biomechanical properties of subchondral bone, promote subchondral bone loss, and reduce subchondral bone quality in early OA mice.
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Damage of hyaline cartilage species such as nasoseptal or joint cartilage requires proper reconstruction, which remains challenging due to the low intrinsic repair capacity of this tissue. Implantation of autologous chondrocytes in combination with a biomimetic biomaterial represents a promising strategy to support cartilage repair. The aim of this work was to assess the viability, attachment, morphology, extracellular matrix (ECM) production of human articular and nasoseptal chondrocytes cultured in vitro in porous poly(l-lactic) (PLLA) scaffolds of two selected pore sizes (100 and 200 μm). The PLLA scaffolds with 100 and 200 μm pore sizes were prepared via ternary thermally induced phase separation (TIPS) technique and analyzed using scanning electron microscopy (SEM). Articular and nasoseptal chondrocytes were seeded on the scaffold and cultures maintained for 7 and 14 days. Live/dead staining, (immuno-)histology and gene expression analysis of type II, type I collagen, aggrecan and SOX9 were performed to assess scaffold cytocompatibility and chondrocyte phenotype. The majority of both chondrocyte types survived on both scaffolds for the whole culture period. Hematoxylin-eosin (HE), alcian blue (visualizing glycosaminoglycans) stainings, immunoreactivity and gene expression of ECM proteins and cartilage marker (type II, I collagen, aggrecan, SOX9) of the chondrocyte scaffold constructs indicated that the smaller pore dimensions promoted the differentiation of the chondrocytes compared with the larger pore size. The present work revealed that the scaffold pore size is an important factor influencing chondrocyte differentiation and indicated that the scaffolds with 100 μm pores serve as a cytocompatible basis for further future modifications.
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Article
  • Nov 2016
Natural and synthetic cross-linked polymers allow the improvement of cytocompatibility and mechanical properties of the individual polymers. In osteochondral lesions of big size it will be required the use of scaffolds to repair the lesion. In this work a borax cross-linked scaffold based on fumarate-vinyl acetate copolymer and chitosan directed to osteochondrondral tissue engineering is developed. The cross-linked scaffolds and physical blends of the polymers are analyzed in based on their morphology, glass transition temperature, and mechanical properties. In addition, the stability, degradation behavior, and the swelling kinetics are studied. The results demonstrate that the borax cross-linked scaffold exhibits hydrogel behavior with appropriated mechanical properties for bone and cartilage tissue regeneration. Bone marrow progenitor cells and primary chondrocytes are used to demonstrate its osteo- and chondrogenic properties, respectively, assessing the osteo- and chondroblastic growth and maturation, without evident signs of cytotoxicity as it is evaluated in an in vitro system.
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Poly (L-Lactic Acid) Porous Scaffold-Supported Alginate Hydrogel with Improved Mechanical Properties and Biocompatibility
Article
  • Sep 2016
  • INT J ARTIF ORGANS
Purpose: Polymer porous scaffolds and hydrogels have been separately employed and explored for a wide range of applications including cell encapsulation, drug delivery, and tissue engineering. Methods: In this study, a three-dimensional poly (L-lactic acid) (PLLA) scaffold with interconnected and homogeneously distributed pores was fabricated to support the alginate hydrogel (Alg). The gels were filled into the porous scaffold, which acted as an analogue of native extracellular matrix (ECM) for entrapment of cells within a support of predefined shape. The mechanical strength of the composite scaffold was characterized by compression testing. The chondrocyte behavior in the scaffold was determined by inverted microscopy, scanning electron microscopy (SEM) and MTT viability assay. The repair efficiency of such a composite scaffold was further investigated in dog spinal defects by histological evaluation after implantation for 4 weeks. Results: Results showed that the composite scaffold possessed superior mechanical properties and hierarchical porous structure in comparison to pure Alg. Cell culture revealed that the cells presented a specific cartilage status in the composite scaffold in line with higher adherence and proliferation ratio. The histological analyses suggested that the composite scaffold substantially promotes its integration in the host tissue accompanied with a low inflammatory reaction and new tissue formation. Conclusions: The method thus provides a useful pathway for scaffold preparation that can simultaneously achieve suitable mechanical properties and good biocompatibility.
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One-Step Cartilage Repair Technique as a Next Generation of Cell Therapy for Cartilage Defects: Biological Characteristics, Preclinical Application, Surgical Techniques, and Clinical Developments
Article
  • Apr 2016
  • ARTHROSCOPY
Purpose: To provide a comprehensive overview of the basic science rationale, surgical technique, and clinical outcomes of 1-step cartilage repair technique used as a treatment strategy for cartilage defects. Methods: A systematic review was performed in the main medical databases to evaluate the several studies concerning 1-step procedures for cartilage repair. The characteristics of cell-seed scaffolds, behavior of cells seeded into scaffolds, and surgical techniques were also discussed. Clinical outcomes and quality of repaired tissue were assessed using several standardized outcome assessment tools, magnetic resonance imaging scans, and biopsy histology. Results: One-step cartilage repair could be divided into 2 types: chondrocyte-matrix complex (CMC) and autologous matrix-induced chondrogenesis (AMIC), both of which allow a simplified surgical approach. Studies with Level IV evidence have shown that 1-step cartilage repair techniques could significantly relieve symptoms and improve functional assessment (P < .05, compared with preoperative evaluation) at short-term follow-up. Furthermore, magnetic resonance imaging showed that 76% cases in all included case series showed at least 75% defect coverage in each lesion, and 3 studies clearly showed hyaline-like cartilage tissue in biopsy tissues by second-look arthroscopy. Conclusions: The 1-step cartilage repair technique, with its potential for effective, homogeneous distribution of chondrocytes and multipotent stem cells on the surface of the cartilage defect, is able to regenerate hyaline-like cartilage tissue, and it could be applied to cartilage repair by arthroscopy. Level of evidence: Level IV, systematic review of Level II and IV studies.
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