Preparation of PLGA-collagen hybrid scaffolds with controlled pore structures for cartilage tissue engineering

Nur Rofiqoh Evin Putri ,Xiuhui Wng ,Ying Chen ,Xiomeng Li ,Noki Kwzoe ,Guoping Chen,*

a Research Center for Functional Materials,National Institute for Materials Science,1-1 Namiki,Tsukuba,Ibaraki,305-0044,Japan

b Department of Materials Science and Engineering,Graduate School of Pure and Applied Sciences,University of Tsukuba,1-1-1 Tennodai,Tsukuba,Ibaraki,305-8577,Japan

c Departement of Chemical Engineering,Faculty of Engineering,Universitas Gadjah Mada,Jl.Grafika No.2,Kampus UGM,Yogyakarta,55281,Indonesia

Keywords:Hybrid scaffolds Porous scaffolds PLGA Collagen Cartilage tissue engineering

ABSTRACT Scaffolds used for cartilage tissue engineering should have high mechanical strength and well-controlled pore structure to provide suitable microenvironments for functional tissue regeneration.In this study,hybrid scaffolds which had open and interconnected pore structure and high mechanical property were prepared by hybridization of PLGA mesh and collagen using ice particulates as porogen templates.Embossing ice particulates template was used to form open pore structures on the scaffold surfaces.Free ice particulates were used to generate interconnected bulk pores in the scaffolds.Hybridization with PLGA mesh provided the scaffolds with high mechanical property.Bovine articular chondrocytes were cultured in the hybrid scaffolds.The unique pore structures facilitated the homogeneous distribution of chondrocytes and cartilaginous matrices throughout the scaffolds.Subcutaneous implantation demonstrated cartilage-like tissue regeneration.The hybrid scaffolds should have a high potential for cartilage tissue engineering.

1.Introduction

Articular cartilage is a thin and smooth tissue which encloses the surface of diarthrodial joints [1].It has an important function in our body to distribute the load and reduce friction during motion [2].Compared to other tissues,cartilage is avascular.It does not have any blood vessels which are needed for nutrient transport during tissue regeneration [3–5].If there is a defect within cartilage tissue,it is difficult to regenerate by itself.Many treatments have been used to heal cartilage damage,such as medicine,microfracture technique,allografting and autografting.Even though these treatments have some effects to assist the regeneration of damaged cartilage tissue,there are also some limitations.Medicine treatment requires long term recovery and only regenerates damaged tissue to normal function partially [6].Microfracture technique can pull out the subchondral bone marrow progenitor cells and repair the defects,but it is only suitable for small defects[7].Surgical strategies,such as cartilage autograft and allograft,are still restricted by donor shortage and the possibility of immune rejection [8–10].Cartilage tissue engineering has been developed and demonstrated as one alternative method to overcome the limitations of these treatments [11–15].

In cartilage tissue engineering,porous scaffolds,extracellular matrices and hydrogels have been used to control cell functions and to guide new tissue regeneration [16–27].3D porous scaffolds have important functions to provide desirable porous structures for cell accommodation and activity [16–18].Many methods have been used to prepare porous scaffolds,such as phase separation [19],gas-forming[20],freeze-drying [21],solvent casting/particulate leaching [22] and additive manufacturing (AM) [23].These methods have problems related to the remaining toxic organic solvents,closed pore structures and undesirable skin layer formation [25,26].AM technique can form complex architecture of scaffolds,but it has high cost and limited materials problem [27].Beside well-controlled pore structures,the strong mechanical property is required to maintain necessary spaces for cell proliferation and matrix secretion till new tissue regeneration.Even though natural polymer-based scaffolds,such as collagen and gelatin scaffolds,provide many advantages including high bioactivity and good biocompatibility,they have poor mechanical property.On the other hand,poly(lactide-co-glycolide) (PLGA) mesh,which has been approved for medical applications by the United States Food and Drug Administration (FDA),has excellent mechanical property.It has been used to reinforce porous scaffolds of naturally derived polymers and extracellular matrices [28–32].

Therefore,in this study,3D porous hybrid scaffolds were prepared by hybridization of PLGA mesh and collagen sponge by using ice particulates to control their pore structures.Ice particulates could serve as nuclei to initiate formation of new ice crystals.The ice particulates and new ice crystals could result in the formation of interconnected pore structures[33,34].Ice particulates could be easily eliminated by freezedrying process.By using uniformly sized ice particulates as porogen templates to control the bulk pore structure of the scaffolds,homogeneous pore structures with good interconnectivity could be formed.Embossing ice particulates templates could help the formation of open porous structures on the surfaces of scaffolds [35–37].In addition,PLGA mesh could enhance the mechanical property of the hybrid scaffolds.The hybrid scaffolds were used for 3D culture of bovine articular chondrocytes (BACs) and subcutaneously implanted in nude mice to confirm their promotive effects on cartilage tissue engineering.

2.Materials and methods

2.1.Scaffold preparation

The schematic preparation procedure is shown in Fig.1.At first,a 2.0(w/v) % aqueous solution of collagen was prepared by dissolving porcine type I collagen (Nitta Gelatin,Japan) in a mixture solution of ethanol and acetic acid (10:90 (v/v),pH 3.0) at 4 °C.The mixture solution of ethanol and acetic acid was used to decrease the freezing point of collagen aqueous solution.Free ice particulates were prepared by spraying Milli-Q water into liquid nitrogen.The ice particulates were then sieved at a low-temperature chamber (ESPEC,Japan) at -15 °C through two sieves having a mesh size of 425 and 500 μm to obtain ice particulates sizing between 425 and 500 μm.The ice particulates were then collected and put in closed glass bottles in the low-temperature chamber and kept at -5 °C for 2 h.Embossing ice particulates on a surface were prepared by using an ultrasonic humidifier(Iori-Mu,GRE-01,ICC Co.,Ltd.,Japan).Glass plates were covered with perfluoroalkoxy (PFA) film and then settled in a moisture chamber of the ultrasonic humidifier for 6 min to form hemispheric water droplets.The glass plates bearing water droplets were then placed in a deep freezer at-80°C for 1 h to form the embossing ice particulates on glass surfaces.

A knitted polyactin 910 mesh (90:10 copolymer of glycolide and lactide (PLGA),Ethicon Inc.,Somerville,USA) was used for the hybridization.The ice particulates,collagen aqueous solution,two embossing ice particulate templates,two silicone molds (thickness:2 mm),PLGA mesh and all other materials were put in a -5 °C lowtemperature chamber for 2 h to balance the temperature.The following manipulations were conducted in the-5°C low-temperature chamber.The free ice particulates were added to the 2.0 (w/v)% collagen aqueous solution at a ratio of 50:50 (w/v) and mixed well using a steel spoon.The 1st silicone mold was settled onto one embossing ice particulate template and then the free ice particulates/collagen mixture solution was poured into the silicone mold.After the surface of the free ice particulates/collagen mixture solution was flattened with a steel spatula,the PLGA mesh was placed on the silicone mold.Afterward,the 2nd silicone mold was exactly settled on the 1st mold and filled with the free ice particulates/collagen mixture solution.The surface of the mixture solution was flattened and covered with another embossing ice particulate template.The whole construct was kept at -12 °C for 4 h for ice crystallization and further frozen at -80 °C for 6 h.The frozen construct was then detached from the silicone molds and glass plates and lyophilized for 48 h.After lyophilization,the scaffolds were crosslinked with 50 mM 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide(EDC,Peptide Institute,Inc.,Japan) and 20 mM N-hydroxysuccimide(NHS,Wako Pure Chemical Industries,Ltd.,Japan) in an 80% (v/v)ethanol aqueous solution at room temperature under shaking (～20 rpm) for 8 h.After cross-linking,the scaffolds were washed 3 times with Milli-Q water and immersed in 0.1 M glycine aqueous solution to block remaining activated residues.The scaffolds were finally washed with Milli-Q water for 6 times.The as-prepared PLGA-collagen hybrid scaffolds were denoted as IC scaffolds.For comparison,normal PLGAcollagen hybrid scaffolds were prepared by the above-described method without free ice particulates and embossing ice particulate templates(denoted as CC scaffolds).

2.2.Characterization of scaffolds

Fig.1.Preparation scheme of PLGA-collagen hybrid scaffolds with controlled pore structures (IC scaffolds).

Pore structures of the scaffolds were analyzed by a JSM-5610 Scanning Electron Microscope (SEM;JEOL,Tokyo,Japan).Scaffolds were sectioned in both horizontal and vertical directions using a razor blade and then coated with platinum by a sputter coater (E-1030,Hitachi,Japan) before observation.Pore size was analyzed by measuring the diameters of pores from 4 SEM images of each type scaffold with an ImageJ software (ImageJ2,NIH,USA) [34].

Static compression and tensile tests were conducted to measure mechanical strength of the scaffolds.The scaffolds used in both tests were in a hydrated state.For the compression test,the constructs were punched into cylindrical discs (Φ6 mm × H4 mm) using a biopsy punch.They were then hydrated in phosphate buffered saline (PBS)solution for 2 h.Compression force was applied to the scaffolds in a direction perpendicular to the PLGA mesh at a rate of 0.1 mm/s.The stress-strain curves were obtained with a texture analyzer (TA.XTPlus,Texture Technologies Corp.,USA).Young's modulus was calculated from the slope of the linear elastic region of the stress-strain curve.For tensile test,the scaffolds were cut into bars with a size of 30 mm×5 mm×4 mm.The two sides of the scaffold bars were fixed on the clips of the tensile test machine.Forces were applied in a direction parallel to the PLGA mesh.The samples were stretched at a rate of 0.1 mm/s until rupture.The ultimate tensile force of each scaffold was determined at the peak of the stress-strain curve.

For porosity measurement,the cylindrical discs of each sample (Φ6 mm×H4 mm)were used.The porosity of each sample was calculated from the weight of water filling the void spaces in the scaffolds by following formula [38,39].

Where Vporeis the pores volume,V is the scaffold volume,W1is the weight of dry scaffold,W2is the weight of hydrated scaffold with water,and ρ is the density of water.Triplicate samples were used for the measurements.

2.3.In vitro cell culture

Bovine articular chondrocytes (BACs) were isolated from bovine articular cartilage by collagenase digestion.The isolated BACs were sub-cultured with high-glucose Dulbecco's Modified Eagle's Medium(D6546,Sigma Aldrich Co.,USA) in 175 cm2tissue culture flasks.The culture medium contained 4500 mg/L glucose and were supplemented with 10% fetal bovine serum,4 mM glutamine,100 U/ml penicillin,100 μg/ml streptomycin,0.1 mM nonessential amino acids,0.4 mM proline,1 mM sodium pyruvate and 50 μg/ml ascorbic acid.The subcultured cells after two passages (P2) were harvested and then suspended in culture medium to get a cell suspension solution of 2.5×107cells/mL for cell seeding.The cylindrical discs(Φ6 mm×H4 mm)of IC and CC scaffolds were sterilized with ethylene oxide gas.The sterilized scaffolds were pre-wetted with a culture medium.After removing the culture medium from the scaffolds using sterilized Kimwipes,100 μl of BACs suspension solution was seeded onto one side of the scaffolds and cultured at 37 °C for 3 h.After that,the scaffolds were turned upside down and another 100 μl of BACs suspension solution was seeded.They were then cultured at 37 °C for another 3 h.After that,the cell-seeded scaffolds were transferred to new 24-well plates and fresh culture medium was added.After culture for 1 day,the cells/scaffold constructs were moved to 75 cm2tissue culture flasks and cultured under shaking (60 rpm) for 1 week.

2.4.Cell distribution in scaffolds

After 1 day of culture,the cells/scaffold constructs were washed with PBS,fixed in 10%neutral buffered formalin for 2 days,dehydrated and embedded in paraffin.They were then sectioned to get the vertical cross-sections at a thickness of 7 μm.The cross-sections were deparaffinized and cell nuclei were stained with 2 μg/ml 4’,6-diamidino-2-phenylindole dihydrochloride solution (DAPI,Dojindo Molecular Technilogies,Inc.,Japan).The stained samples were observed with a fluorescence microscope (Olympus,Japan).

2.5.In vivo implantation

All the animal experimental procedures were approved by the Animal Experiments Committee of the National Institute for Materials Science and the experiment was conducted according to the committee guidelines.After 1 week of in vitro culture,the cells/scaffold constructs were implanted subcutaneously into the back of 6-week-old athymic nude mice.Each mouse was implanted with four scaffolds.After implantation for 8 weeks,the mice were sacrificed to retrieve the implants.The gross appearance of the harvested implants after 8 weeks of implantation was observed with an OLYMPUS DP22 light microscope.

2.6.Histological and immunohistochemical staining

The implants were fixed in 10%neutral buffered formalin for 2 days at room temperature.They were dehydrated,embedded in paraffin,and sectioned to get the vertical cross-sections at a thickness of 7 μm.The vertical cross-sections were stained with hematoxylin and eosin (HE)and safranin O.The cross-sections were also immunohistochemically stained with anti-collagen type II antibody.The deparaffinized sections were incubated with proteinase K for antigen retrieval for 10 min,peroxidase blocking solution for 5 min and 10%goat serum solution for 30 min.Afterward,they were incubated with rabbit polyclonal anticollagen type II(in a 1:100 working dilution)(Millipore®)for 2 h.After washing,a peroxidase-labeled polymer-conjugated second antibody(Envision+System-HRP(DAB))was applied for 1 h.Finally,they were incubated with 3,3′-diaminobenzidine (DAB) for 5 min to develop the color.The stained sections were observed with a light microscope.All procedures were conducted at room temperature.

2.7.Biochemical quantification and compressive test

Quantification of DNA and sulfated glycosaminoglycan (sGAG)amounts was conducted according to the previous report [25].Briefly,after in vitro culture for 1 day and implantation for 8 weeks,the cells/scaffold constructs and implants were washed with pure water,lyophilized and digested with 400 μg/ml papain solution containing 5 mM L-cysteine and 5 mM EDTA in 0.1 M phosphate buffer at pH of 6.0.DNA amount was measured with a DNA Quantitative Kit(Sigma,USA)and a spectrofluorometer (FP-6500;JASCO,Japan).The papain solution was also used for the measurement of sGAG content by using a BlyscanTMSulfated Glycosaminoglycan Assay Kit (Biocolor,Carrickfergus,UK).The compression strength of the 8-week implants was measured by the compression test as described above.Triplicate samples were used for all these measurements.

2.8.Gene expressions of the cartilaginous matrix proteins

Expression of cartilaginous genes encoding bovine-type II collagen and aggrecan in the cells/scaffold constructs was evaluated with realtime polymerase chain reaction (RT-PCR).The 8-week implants were rinsed with PBS buffer,frozen at -80 °C,crushed into powder by an electric crusher and dissolved in Sepasol solution (Nacalai Tesque) to isolate RNA from the cells.Afterward,the RNA was converted to cDNA by MuLV reverse transcriptase(Applied Biosystems,USA).RT-PCR was performed to amplify glyceraldehyde-3-phosphate dehydrogenase(GAPDH),collagen type II(Col2a1),and aggrecan(Acan).The reactions were conducted in 0.2 μlcDNA,18 mM forward/reverse primers,5 mM PCR probe and TaqMan® Fast Advanced Master Mix (Applied Biosystems,Thermo Fisher Scientific) in duplicate.The reactions were run for 40 cycles using a Quant Studio 3 Real-Time PCR System(Applied Biosystems).Triplicate samples were used for RT-PCR measurement.The primer and probe sequences are shown in Table 1 [40].

Table 1 The primer and probe sequences for GADPH,bovine-type II collagen and aggrecan.

Table 2 Pore size and porosity of IC and CC scaffolds.Data represent mean ± S.D.(n=3).

2.9.Statistical analysis

All statistical analyses were performed by using a one-way analysis of variance (ANOVA) with Tukey's post hoc test for multiple comparisons to check the significant difference among the samples.Data are presented as the mean ± standard deviation (SD).A significant difference was considered when the p-value was less than 0.05.The data are indicated with ＊ for p ＜ 0.05,＊＊ for p ＜ 0.01 and ＊＊＊ for p ＜0.001.

3.Results and discussion

3.1.Characteristics of ice particulates and hybrid scaffolds

The free ice particulates had spherical morphology with a size in a range of 425–500 μm (Fig.2a).The embossing ice particulates on the template surface had hemispherical morphology with an average diameter of 442 ± 10 μm (Fig.2b).The gross appearance of IC and CC scaffolds after punching into cylindrical discs is shown in Fig.2c.Both hybrid scaffolds showed white color.

The microstructures of IC and CC scaffolds were observed by SEM(Fig.3).The IC scaffolds had many spherical large pores on their surfaces.The surface pores were open and interconnected with the underlying bulk pores.However,the surface of CC scaffolds had randomly shaped pores and most of the surface pores were closed.The images of both horizontal and vertical cross-sections showed the interconnected spherical large pore structures in the inner bulk areas of IC scaffolds.However,the pores in CC scaffolds were irregular and only partially interconnected.The embossing ice particulate template should result in the open and spherical pores on the surface of IC scaffolds and the free ice particulates should contribute to the interconnected spherical large pores in the bulk areas of IC scaffolds [41,42].Without using of embossing ice particulates template and free ice particulates,the CC scaffolds did not have such porous structures.The PLGA mesh was observed at the center of both CC and IC scaffolds.As a previous study shows that PLGA mesh can be placed at the center or one side of the collagen sponge [28].PLGA mesh was placed at the center of the IC hybrid scaffolds in this study to allow both sides of the scaffolds could have open surface pore structures for cell seeding.The vertical crosssection images showed that the PLGA mesh was integrated well with collagen sponge layers.

Fig.2.Photomicrographs of free ice particulates (a) and embossing ice particulates template (b).Gross appearance of IC and CC scaffolds (c).Scale bar in a and b=500 μm.Scale bar in c=5 mm.

Fig.3.SEM images of surfaces,horizontal and vertical cross-sections of IC and CC scaffolds.Left columns are SEM images at low magnification and right columns are images at high magnification.The green squares,blue arrows,red circles and yellow circles indicate magnification regions,PLGA mesh,large spherical pores and interconnecting pores,respectively.Scale bar at low magnification=500 μm.Scale bar at high magnification=100 μm.

The pore size and porosity of IC and CC scaffolds are shown in Table 2.Two types of pores in IC scaffolds were used for the measurements of pore size.One type was the spherical large pores as shown in red in Fig.3.These pores were formed as a negative replica of the free ice particulates after freeze-drying.Another type of pores was the small pores that were the negative replica of new ice crystals that were formed during the freezing process of the mixture solution.The small pores were formed among the large pores,making the large pores interconnected.The size of spherical large pores was in accordance with the size of free ice particulates.However,the CC scaffolds showed limited pore structures with a smaller size.Some of the pores were open while some were closed.The porosity of both IC and CC scaffolds was higher than 98%.The high porosity and good interconnected pore structure of IC scaffolds should be beneficial for cell penetration and distribution.

3.2.Mechanical properties of hybrid scaffolds

The compression and tensile strength of IC and CC scaffolds were measured at hydrated states (Fig.4).The compressive modulus and ultimate tensile force of IC scaffolds were a little higher than those of CC scaffolds.However,there was no significant difference.The pore structure did not significantly affect the mechanical properties of the hybrid scaffolds.The PLGA mesh was not used for the compression test but only used for the tensile test because it was very thin (～200 μm thick).The ultimate tensile force of IC and CC scaffolds was almost the same as that of PLGA mesh.The results indicated that the PLGA mesh could provide hybrid scaffolds with high tensile strength.The results were similar to the previous report showing that mechanically strong synthetic polymers can be used to strengthen the porous structures of naturally derived polymers such as collagen and gelatin[18,28,29,32].

3.3.Cell distribution in hybrid scaffolds

BACs were seeded and cultured in IC and CC scaffolds.Cell distribution was investigated by nucleus staining (Fig.5).BACs showed homogeneous distribution in the IC scaffolds,which should be due to the open surface pore and interconnected bulk pore structures of IC scaffolds.However,BACs were predominantly distributed on the surface areas of CC scaffolds.The poor interconnectivity of CC scaffolds limited cell diffusion,resulting in most of the seeded cells on the scaffold surfaces.

3.4.Gross appearance,histological and immunohistochemical analyses of engineered cartilage tissue

After in vitro culture for 1 week,the cells/scaffold constructs were subcutaneously implanted in the back of nude mice for 8 weeks.The gross appearance of the implants showed a cartilaginous appearance with glossy white in color (Fig.6a and b).

HE staining showed homogenous cell distribution and deposition of extracellular matrix (ECM) throughout the IC scaffolds (Fig.6c and d).On the other hand,cells in CC scaffolds showed heterogeneous distribution,where most of the cells were distributed on the surface leading to many voids in the inner bulk areas of the scaffolds.Safranin O staining showed a more uniform formation of cartilaginous matrices in IC scaffolds than that in CC scaffolds (Fig.6e and f).Many void spaces were observed in CC scaffolds.The typical marker protein,collagen type II,was immunohistochemically stained to show cartilage tissue formation (Fig.6g and h).The results showed that IC scaffolds promoted a more homogeneous formation of collagen type II than did CC scaffolds.Collagen type II in CC scaffolds was inhomogeneous with some void spaces remaining inside the scaffolds.These results indicated that the open and interconnected pore structures in IC scaffolds facilitated not only homogeneous cell distribution but also homogeneous cell growth and homogeneous deposition of cartilaginous matrices.The interconnected pore structure inside IC scaffolds could enhance cell penetration and migration throughout the scaffolds,thus resulting in more homogeneous cell growth,ECM deposition and tissue regeneration than those in CC scaffolds.

Fig.4.Compression modulus of IC and CC scaffolds(a).Ultimate tensile force of IC and CC scaffolds and PLGA mesh.All the scaffolds were tested at a hydrated state.Data represent mean ± S.D.(n=3).

Fig.5.Cell distribution in IC and CC scaffolds after in vitro culture for 1 day.Cell nuclei were stained with DAPI.Scale bar=300 μm.

3.5.DNA and sGAG quantification and mechanical properties of engineered cartilage tissue

DNA amount in both IC and CC scaffolds increased with culture time(Fig.7a).BACs proliferated in both scaffolds.After in vitro culture for 1 day,there was no significant difference in DNA amount between IC and CC scaffolds,demonstrating the same amounts of cells were seeded in both scaffolds.After in vitro culture for 1 week and in vivo implantation for 8 weeks,the DNA amount in IC scaffolds was higher than that in CC scaffolds.The sGAG synthesis in both scaffolds increased with culture time and showed the same pattern as that of DNA content (Fig.7b).After implantation for 8 weeks,the amount of sGAG in IC scaffolds was higher than that in CC scaffolds.The results indicated that BACs proliferated more quickly and secreted more cartilaginous matrices in IC scaffolds than did in CC scaffolds.

The compression modulus of engineered cartilage tissue was evaluated by the compression test (Fig.7c).Compared to the hydrated scaffolds before cell seeding,the compression modulus of implants increased significantly after 8 weeks of implantation.The compression modulus of cartilage tissue formed in IC scaffolds was significantly higher than that formed in CC scaffolds.

3.6.Gene expression of engineered cartilage tissue

The expression of cartilaginous marker genes after in vivo implantation for 8 weeks was analyzed by RT-PCR (Fig.8).The relative expression level of gene encoding collagen type II showed no significant different between IC and CC scaffolds.However,the expression level of aggrecan in IC scaffolds was significantly higher than that in CC scaffolds.

All the results indicated that the open surface pores and interconnected bulk pores in IC scaffolds resulted a homogenous distribution of BACs,homogenous cell growth and deposition of cartilaginous matrices throughout the scaffolds.The homogenous distribution of cells and matrices facilitated cell-cell and cell-ECM interaction throughout the IC scaffold,therefore inducing a high level of gene expression and homogeneous tissue regeneration.The homogenously regenerated tissue had high mechanical strength.On the other hand,the CC scaffolds prepared without embossing ice particulates template or free ice particulates had poor pore interconnectivity,therefore resulting in predominant cell distribution on scaffold surfaces.Although BACs on the surfaces of CC scaffolds could proliferate and produce cartilaginous matrices,the cells and matrices were mainly distributed on the surfaces,resulting in tissue formation on skin layers and leaving some of the central areas void.The heterogeneous distribution of cells and cartilaginous matrices in CC scaffolds could explain the significantly lower mechanical property of regenerated cartilage tissue.It has been reported that homogenous cell distribution can facilitate cell-cell interaction and thus facilitate cartilage tissue regeneration [43].The IC scaffolds showed a superior effect on cartilage tissue regeneration to the CC scaffolds.Hybridization of PLGA mesh and collagen with ice particulates template technique could improve the mechanical property and pore structure of the scaffolds.The IC scaffolds had high mechanical property and well-controlled pore structures that facilitated cartilage tissue regeneration.The introduction of a mechanically strong skeleton in mechanically weak scaffolds such as collagen and gelatin has been reported to strengthen their mechanical properties [29–32,44,45].The PLGA mesh in IC and CC scaffolds was introduced in the center of the scaffolds and integrated well with the collagen layers,which contributed to the high mechanical properties of IC and CC scaffolds.The well-controlled pore structures and high mechanical properties of IC scaffolds facilitated homogenous cell distribution and maintenance of spaces for tissue regeneration.The IC scaffolds should be useful for cartilage tissue engineering.

Fig.6.Gross appearance (a,b),HE staining (c,d),Safranin O staining (e,f) and immunohistochemical staining of collagen type II (g,h) of the implants after implantation for 8 weeks.The inserted images indicate the whole cross-sections of implants.Scale bar in a and b=3 mm.Scale bar in c-h=500 μm.

Fig.7.DNA amount(a)and GAG content(b)of cells/scaffold constructs after in vitro culture for 1 day and 1 week and in vivo implantation for 8 weeks.Compression modulus (c) of hydrated scaffolds without cells and cells/scaffold construct (implant) after implantation for 8 weeks.Data represent mean ± S.D.(n=3).＊,p ＜0.05;＊＊,p ＜0.01.

Fig.8.Gene expression of aggrecan (a) and type II collagen (b) of implants after implantation for 8 weeks.Data represent mean ± S.D.(n=3).Significant difference:＊＊p ＜0.01.

4.Conclusions

PLGA-collagen hybrid(IC)scaffolds were prepared by hybridization of PLGA mesh with collagen sponge layers which pore structures were controlled by free ice particulates and embossing ice particulates template.The IC scaffolds had high mechanical strength and well-controlled pore structures of open surface pores and interconnected bulk pores.The open and interconnected pore structures of IC scaffolds facilitated the homogenous distribution of bovine articular chondrocytes and cartilaginous matrices.The IC scaffolds promoted cell proliferation,cartilaginous gene expression and regeneration of cartilage-like tissue with high mechanical property.The IC scaffolds hold high potential for cartilage tissue engineering applications.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This research was supported by JSPS KAKENHI Grant Number 19H04475.

Appendix A.Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.pnsc.2020.07.003.