In Vitro and In Vivo Evaluations of Loofah Micro- and PHBV Nano-Fiber Integrated Hydrogel Scaffolds for Meniscus Regeneration

doi:10.21203/rs.3.rs-4947320/v1

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In Vitro and In Vivo Evaluations of Loofah Micro- and PHBV Nano-Fiber Integrated Hydrogel Scaffolds for Meniscus Regeneration

https://doi.org/10.21203/rs.3.rs-4947320/v1

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Meniscus is a tissue that has vital properties for knee stabilization, shock absorption, axial load distribution, joint lubrication, and nutrition of articular cartilage. This study aims to produce loofah micro- and poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) nano-fiber integrated collagen and chitosan polymer-based composite hydrogel scaffolds crosslinked with three different concentrations of genipin (0.1, 0.3, and 0.5%) for meniscus regeneration. The scaffold crosslinked with 0.3% genipin, which exhibites the highest compression strength with high water absorption and damping capacities, is chosen as the optimal scaffold for further in vitro and in vivo analyses. In vitro biocompatibility studies are conducted by using rabbit bone marrow-derived mesenchymal stem cells. Accordingly, the composite hydrogel scaffold is found to be non-toxic, and capable of promoting cell adhesion and proliferation as well as collagen immunopositive, especially for type II collagen. In vivo analysis is performed by using 24 adult male New Zealand rabbits in three groups (empty defect, cell-free and cell-laden implanted scaffolds) with a standardized meniscus regeneration model. In conclusion, the cell-laden scaffold implanted group shows better meniscal healing based on the post-implantation biomechanical, histological, immunohistochemical, and Micro-CT evaluations.

Hydrogel composite scaffold

mesenchymal stem cells

meniscus tissue engineering

rabbit meniscus regeneration

A crescent-shaped, concave, fibrocartilaginous meniscus tissue exists between the knee joint's tibial plateau and femoral condyle [1]. This tissue stabilizes the knee, absorbs shock, and distributes axial loads [2]. Meniscus tears have increased to approximately 60 per 100,000 population mostly among the people who are involved in sports activities. The meniscus tear can be diagnosed by using a simple radiologic technique, Magnetic Resonance Imaging (MRI). If those tears are left without treatment, meniscus injury leads to knee pain and osteoarthritis [3]. Unfortunately, the meniscus has a poor self-healing capacity due to its limited blood supply. Therefore, the tissue engineering approach with the combination of cells, scaffolds, and growth factors is becoming promising for meniscus repair. This technique deals not only with the regeneration using engineered constructs but also cell-matrix interactions. Meniscus scaffolds should be three-dimensional biomimetic porous constructs that allow cell migration and proliferation without cytotoxicity. The engineered scaffolds should trigger fibrocartilage-like cell growth for constructing meniscus tissue [4].

Mesenchymal stem cells (MSCs) are attractive cell sources in tissue engineering applications. They can be easily isolated from many sources such as; adipose tissue, bone marrow, synovium, umbilical cord, amniotic fluid, periosteum, etc. For the last two decades, the potential of MSCs isolated from different sources was investigated in many studies, and their advantages/disadvantages were determined [5]. These cells can be defined easily by using flow cytometry analysis with the positive expression of CD73, CD90, and CD105, whereas negative to the hematopoietic and endothelial markers of CD34, CD45, and HLA-DR [6]. They also have anti-inflammatory potential with the ability of self-renewal and differentiation [7]. In our previous studies, meniscus scaffolds were successfully produced, and in-vitro biocompatibility studies were conducted by using these multipotent stem cells. MSC-seeded meniscus scaffolds have been found to trigger the deposition of type I and especially type II collagen [8, 9].

Meniscus is mostly composed of type I and II collagen (the wet weight is about 22%), glycosaminoglycans (GAGs) such as chondroitin sulfate (the wet weight is about 0.8%), and water (72%). Negatively charged proteoglycans attract the counter ions and water to the meniscus, and the tissue becomes viscoelastic and absorbs compressive loads. Whereas, the circumferentially arranged collagen bundles provide tensile strength. This anisotropic arrangement of collagen microstructure is important for the meniscus biomechanics [2]. The tensile modulus of menisci was previously described in the literature, which was given in the range of 40–300 MPa circumferentially, and 10–30 MPa radially [10].

Chitosan is a linear polysaccharide produced by the deacetylation of chitin which is mainly obtained from the shells of shrimps and crabs. Chitosan is a widely used polymer in tissue engineering applications due to its structural similarity with GAGs and anti-bacterial properties [11]. On the other hand, collagen is the most abundant protein in the human body which is categorized into almost 28 subtypes. Types I, II, and III collagen are widely distributed in tissues including bone, cartilage, skin, and other connective tissues. Since collagen can provide better biomimicry properties, researchers have preferred to use collagen in tissue engineering applications [12].

Our research group previously prepared a collagen-chitosan hydrogel composite scaffold consisting of 3D printed PLA strut and cellulose nanofibers [9] as well as a loofah-reinforced and PHBV nanofiber incorporated chitosan hydrogel composite scaffold for meniscus regeneration [8]. In both studies, micro- and nano-sized topographical features favored MSCs adhesion and spreading. Especially, loofah together with PHBV nanofibers were shown to be a promising reinforcement to regenerate meniscus tissue. Loofah is a fibrous plant named Luffa cylindrica and it was first introduced as a potential porous scaffolding material for bone and cartilage tissue engineering by Cecen, B. [13] and Baysan, G. [14]. Besides, PHBV which is a biodegradable microbial polyester, is another preferred polymer in tissue engineering applications due to its high spinnability in nanofibrous form and biocompatibility [15].

In light of our previous studies, in the present study, novel collagen-chitosan hydrogel composite scaffolds crosslinked with three different concentrations of genipin (0.1, 0.3, and 0.5%) were produced comprising PHBV nanofiber integrated loofah mats for meniscus tissue engineering applications. Scanning electron microscope (SEM) and Fourier-transformed infrared spectrometer (FTIR) were used for the morphological and chemical characterizations of the scaffolds, respectively. The swelling ratio and water content values of the scaffolds were calculated using a swelling test. The scaffolds’ viscoelastic and mechanical properties were determined by dynamic mechanical analysis and compression test, respectively. Furthermore, in vitro biocompatibility analysis was conducted by using rabbit bone marrow-derived mesenchymal stem cells (rMSCs). Cytotoxicity and cell proliferation assessments of the scaffolds were performed using LDH and WST-1 assay kits, respectively. SEM and fluorescence microscopy were used to observe cell attachment and spreading. Besides, histological stainings were examined to observe the cells on the cross-sections of the scaffolds and immunohistochemical analyses were evaluated in terms of collagen type I and II depositions. In addition, the biocompatibility of the scaffolds was further investigated by in vivo analysis performed on 24 adult male New Zealand rabbits in three groups (n = 8) (empty defect, cell-free and cell-laden implanted scaffolds) following a meniscus regeneration model. Finally, post-implantation microcomputer tomography (Micro-CT) imaging, in vivo biomechanical tests, histological and immunohistochemical analyses were evaluated for meniscus regeneration success of the designed scaffolds implanted with or without rMSCs.

2.1. Materials

The loofah plant was supplied from the Turkish Republic of Northern Cyprus. PHBV (PHV content 3 wt%, Mn = 80 kDa) was purchased from Helian Polymers, Netherlands. Chitosan (high molecular weight, deacetylation degree ≥ 75), collagen, and phosphate-buffered saline (PBS) were purchased from Sigma-Aldrich, USA. Genipin was purchased from Wako Chemical, USA. Sodium hydroxide (NaOH) and acetic acid were obtained from Merck, Germany.

For in vitro cell culture studies, a rabbit Mesenchymal Stem Cell line (RBXMX-01001) was obtained from Cyagen, USA. Mesenchymal Stem Cell Growth Medium (MSCM, 7501) was purchased from ScienceCell, USA. StemPro Chondrogenesis Differentiation Kit (A10071- 01), StemPro Osteogenesis Differentiation Kit (A10072-01), and StemPro Chondrogenesis Differentiation Kit (A10070-01) were obtained from Gibco, USA. Phosphate Buffer Saline (PBS) and Fetal Bovine Serum (FBS) were purchased from Cegrogen Biotech. Cytotoxicity analysis was conducted by using a Lactate dehydrogenase (Pierce LDH, Thermo Fisher Scientific) kit. Proliferation analysis was performed by using Ready-to-use Cell Proliferation Colorimetric Reagent (WST-1 from BioVision, K304-2500, USA). Cell attachment studies conducted by using NucBlue™ (DAPI) and Alexa Fluor 594 fluorescence dyes were obtained from Invitrogen. Anesthetics ketamine (Ketax, Vem) and xylazine (Rompun, Bayer) were used for in vivo studies.

2.2. Production of PHBV Nanofiber and Loofah Mat Reinforced Collagen-Chitosan Scaffolds

2.2.1. Preparation of Loofah Mats

Dried natural loofah was peeled off and treated in 2% NaOH solution to remove the noncellulosic contents like hemicellulose, lignin, and wax from the surface of the fibers [16]. Then, they were washed with distilled water and cut in the size of the bottom diameter of the glass bottles in which the scaffolds were produced and dried in a vacuum oven at 60ºC for 24 hours. Finally, loofah mats with an approximate weight of 0.1 g were obtained. The preparation stages of the loofah mats were shown in Fig. 1a-d.

2.2.2. Preparation of PHBV Nanofibers

PHBV solution was prepared by mixing 3% (w/v) PHBV and 0.2% (w/v) triethylammonium chloride (BTEAC) in chloroform at 50°C for 2 hours and then stirring at room temperature for 24 hours. In the wet-electrospinning process, the parameters optimized in our previous study were used [15]. The PHBV solution was spun into a bath filled with an ethanol-water mixture (9:1 v/v) at an applied voltage of 20 kV, flow rate of 2.0 ml/h, and 10 cm working distance. After 15 minutes of spinning, the suspended fibers (Fig. 1e) in the bath were collected and washed with pure water [8].

2.2.3. Preparation of Composite Sponges and Hydrogel Scaffolds

Loofah mats (n = 8, weight = 0.0970 ± 0.0072 g) were placed into wet-electrospun PHBV nanofibers suspended in water and left overnight for integration (Fig. 1f). It was then lyophilized (Telstar LyoQuest-85) at -25°C for 2 days at a pressure of 0.1 mbar (Fig. 1g). The final weight of the PHBV nanofiber integrated loofah mats was 0.1084 ± 0.0050 g. For the preparation of composite sponges, first 1 wt% of chitosan solution was prepared by mixing chitosan in 0.2 M acetic acid solution at 50ºC for 3 hours. A collagen solution with a concentration of 4 mg/ml in 0.02 M acetic acid was used as received. Next, collagen and chitosan solutions were mixed to have 3:1 by weight of collagen:chitosan. Then, the collagen-chitosan mixture was added to the PHBV nanofiber-integrated loofah mats and left overnight. Three-dimensional composite sponges were obtained as a result of the lyophilization process performed at -25°C for 2 days at a pressure of 0.1 mbar. Prepared sponges were washed gradually in ethanol-water series (100%, 80, 60, 40, 20, 0 ethanol by volume) to remove acetic acid.

Genipin was used as the crosslinking agent for hydrogel preparation. Genipin solutions at 0.1%, 0.3%, and 0.5% weight/volume ratios were obtained by dissolving genipin in phosphate-buffered saline (PBS) solution at room temperature. Then, 6 ml of genipin solution was added to composite sponges and allowed to cross-link at room temperature. At the end of 48 hours, all the sponges turned dark green and their covalent cross-linking was completed. The scaffolds were washed with distilled water to remove excess genipin that did not participate in the cross-linking reaction and finally dried by lyophilization (Fig. 1h).

The composite hydrogel scaffolds were coded concerning their genipin concentrations where PL/ChtCol-0 refers to a non-crosslinked scaffold (P = PHBV nanofiber, L = loofah mat, Cht = Chitosan, Col = Collagen). In PL/ChtCol-1, PL/ChtCol-3, and PL/ChtCol-5 scaffolds genipin concentrations were 0.1, 0.3, and 0.5 w/v %, respectively (Table 1).

Table 1

List of the prepared composite hydrogel scaffolds (P = PHBV nanofiber, L = loofah mat, Cht = Chitosan, Col = Collagen)
Scaffold Nomenclature	Genipin Concentrations
(PL/ChtCol-0)	-
(PL/ChtCol-1)	0.1
(PL/ChtCol-3)	0.3
(PL/ChtCol-5)	0.5

2.3. Characterization of the Composite Hydrogel Scaffolds

2.3.1. Morphological Characterization

Morphological characterizations of the scaffolds were performed by SEM (JEOL JSM-6060). All the samples were coated with a thin layer of gold/palladium prior to imaging.

2.3.2. Chemical Characterization

The chemical structure of the scaffolds was determined by FTIR (Perkin Elmer SpectrumBX) at wavenumbers between 4000–650 cm^− 1 with a resolution of 4 cm^− 1 and 20 scans per sample.

2.3.3. Swelling Test

For the swelling test, first, the dry weights of the scaffolds (n = 3) were measured (w₀) and then the scaffolds were incubated in PBS at 37ºC for 24 hours. Afterward, the swollen samples were taken out, and the excess solution was removed with filter paper and weighed again (w_s). The swelling ratios (Eq. 1) and water contents (Eq. 2) of the scaffolds were calculated according to the equations given below.

$$\:Swelling\:ratio\:\left(\%\right)=\left(\frac{{w}_{s}-\:{w}_{0}}{{w}_{0}}\right)\:x\:100$$

$$\:Water\:content\:\left(\%\right)=\left(\frac{{w}_{s}-\:{w}_{0}}{{w}_{s}}\right)\:x\:100$$

2.3.4. Viscoelastic Property Characterization

The viscoelastic properties of the hydrogel composite scaffolds were determined by dynamic mechanical analysis (DMA, TA Instruments Q800) using dynamic frequency scanning mode between 0.1 Hz and 100 Hz at 37ºC in compression mode. The storage (E') and loss moduli (E") of the samples were measured and the tan δ (E"/E') values, which is a measure of the damping capacity, were calculated.

2.3.5. Mechanical Test

The mechanical properties of the scaffolds were characterized by compression testing (Shimadzu AG-X, 5 kN). Cylindrical specimens with a diameter of 7 mm and a thickness of 5 mm were compressed at a constant deformation rate of 0.5 mm/s up to a stress of 80%. All the experiments were performed in four replicates at room temperature. Stress values at 80% strain were recorded as compressive strength.

2.4. In vitro Studies

In vitro cell culture studies were carried out by using rabbit bone marrow-derived mesenchymal stem cell lines (rMSCs) at Dokuz Eylul University, Department of Biomechanics Laboratory. rMSCs were incubated at 37°C and 5% CO₂ for 21 days with stem cell growth medium containing 10% FBS, 1% antibiotic/antimycotic solution, 1% MSC growth supplement, and 200 mM L-glutamine. The medium was changed twice a week and cells were observed with an inverted microscope.

2.4.1. Differentiation capacity of rMSCs

rMSCs were seeded on 6-well plates at a density of 3000 cells/cm² and cells were incubated at 37°C and 5% CO₂ with StemPro chondrogenic, osteogenic, and adipogenic medium, respectively.

Chondrogenic differentiation

rMSCs were incubated with StemPro chondrogenic differentiation medium kit for 21 days. The culture medium was changed twice a week and cells were examined with Alcian blue staining using an inverted microscope.

Osteogenic differentiation

rMSCs were incubated with StemPro osteogenic differentiation medium kit for 21 days. The culture medium was changed twice a week and cells were examined with Alizarin Red staining using an inverted microscope.

Adipogenic differentiation

rMSCs were incubated with StemPro osteogenic differentiation medium kit for 28 days. The culture medium was changed twice a week and cells were examined with Oil Red O staining using an inverted microscope.

When the differentiation studies were completed, in vitro biocompatibility studies were performed. Scaffolds were sterilized by ethylene oxide and then rMSCs were seeded on scaffolds at a density of 5x10⁵ cells/ml for biocompatibility analysis. Scaffolds were incubated at 37°C and 5% CO₂ for 21 days with a stem cell growth medium kit. The culture medium was changed twice a week.

2.4.2. Cytotoxicity analysis

Cytotoxicity analysis was evaluated by using a LDH assay kit on days 3, 7, 10, and 14. Firstly, the culture medium containing lysis solution was incubated at 37°C for 45 minutes and then poured into a 96-well plate with the reaction solution. Samples were incubated at 37°C for 30 minutes and the amount of LDH released into the medium was measured at 490–680 nm wavelength by a spectrophotometer (Synergy HTX).

2.4.3. Cell proliferation of scaffolds

Cell proliferation on scaffolds was investigated by using a WST-1 cell viability assay kit according to the manufacturer’s instructions and measured colorimetrically by a spectrophotometer at 450 nm on days 3, 7, 10, and 14.

2.4.4. Cell attachment on scaffolds

On culture days 14 and 21, rMSCs were observed for cell attachment. Cells were fixed using a 4% paraformaldehyde (v/v) solution. For SEM analysis, cell-scaffold constructs were dehydrated with ethanol solution in graded series (25%, 50%, 75%, 90%, 100%). They were coated with gold/palladium using a sputter coater (Quorum Technologies, SC7620) and investigated by Scanning Electron Microscopy (JEOL JSM-6060). For fluorescence microscope imaging (Olympus IX71), cell attachment on scaffolds was determined by using NucBlue™ (DAPI) and Alexa Fluor 594 dyes for cell nuclei and cytoskeleton, respectively.

2.5. In vivo Studies

24 male, New Zealand White Rabbits (weight, 2.5-3 kg) were obtained from Dokuz Eylul University, Laboratory of Animal Sciences, Izmir, Turkey. In vivo experimentation was performed in three groups (n = 8) with the approval of the Dokuz Eylul University Experimental Animals Ethical Council (Protocol No: 77/2016).

In vivo studies were performed under sterile and aseptic conditions, and in a standardized environment. Rabbits were anesthetized by a mixture of ketamine (35 ml/kg) and xylazine (5 ml/kg) intramuscularly and placed in the supine position. Their right knees were shaved and cleaned with a povidone-iodine solution. A longitudinal 5 cm incision was created from the upper pole of the patella to the tibia tuberosity. The joint was opened with a medial parapatellar approach and the patella was deviated laterally. The medial meniscus was exposed by maximized flexion of the knee joint and a 1.5 mm diameter full-thickness defect was created in the medial meniscus [17].

The first group was a control group and the defect site was left empty. The second group was the material control group for biocompatibility in which a 3 mm thick ethylene oxide sterilized cell-free scaffold was implanted to the defect site with the help of a fibrin glue. The last group was the 3 mm thick rMSC seeded scaffold group implanted to the defect site again with the help of fibrin glue.

After the patella was reduced to its position, the knee joint and skin were closed with 4/0 vicryl and proline suture. The rabbits were kept alive postoperatively for twelve weeks to observe the meniscus regeneration. After that, they were sacrificed with a high-dose anesthetic. Finally, biomechanical, histological, immunohistochemical, and Micro-CT analyses were performed.

2.6. Micro-CT Characterizations

Micro-CT analyses were performed at Ege University, Central Research Test and Analysis Laboratory Application and Research Center (Ege-MATAL). After the 12th week of implantation, the cell-laden and cell-free PL/ChtCol-3 scaffold groups were analyzed by using SCANCO MEDICAL µCT 50 device. Prior to analysis, the knee joints of the rabbits were preserved and the tissues were dissected from the tibia and femur bones. Micro-CT scanning parameters were as follows; 70 kVp energy, 114 µA intensity, 20 µm voxel size, 300 ms integration time, and 0.1 mm Al filter. Thin section and 3-dimensional (3D) analyses were made by using the DICOM program.

2.7. Post-Implantation Biomechanical Studies

The rabbit knees were thawed at RT with distilled water. Each knee was fixed with a custom-made platen, and a stainless steel 3mm diameter indenter was positioned over the defect site without any contact. The compression test was performed at a 10mm/min speed, which stopped at a 3mm displacement (5kN AG-X; Shimadzu, Kyoto, Japan).

2.8. Histological Stainings and Immunohistochemical Analysis

For both in vitro and in vivo studies, Hematoxylin-Eosin (H&E) and Masson Trichrome were used to observe general morphology and the presence of collagen structures in the histological stainings, respectively. In addition, collagen type I and II structures formed in the scaffolds were examined immunohistochemically by the Avidin-Biotin-Peroxidase method [8]. Furthermore, for detailed in vivo investigations, Alcian Blue [18] and Alizarin Red S [19] stainings were used to observe GAG structure and mineralization, respectively.

Prior to histological and immunohistochemical stainings, the scaffolds and in vivo tissues dissected after scarification were fixed with 4 and 10% formalin solution, respectively. Then, they were embedded in paraffin blocks after a routine follow-up study overnight. With the help of a microtome, 5µm sections were taken on polylysine-coated slides and kept in an oven at 60^oC for deparaffinization. The staining of the sections was completed with the help of the previously mentioned dyes. The sections were washed with xylene followed by decreasing alcohol series (96, 70, 50, 25, and 0), and then observed with the microscope.

2.9. Statistical Analysis

The data were analyzed as mean ± standard error of at least three replicates. Statistical analysis was carried out using a one-way variance analysis (ANOVA) test, and Post-hoc Tukey's test was used for multiple comparisons. Statistically significant value was considered as p ≤ 0.05 ^[17].

3.1. Production and Characterization of the PL/ChtCol Scaffolds

For the preparation of composite hydrogel scaffolds, PHBV nanofibers and loofah mats were used as the composite reinforcements (Fig. 1). Thus, in addition to mechanical properties, micro- and nano-sized topographical features that support cellular activities were provided in the structure. The SEM images of the fabricated scaffolds are given in Fig. 2a. The integration of the loofah mats and PHBV nanofibers with the collagen-chitosan matrix was very important in terms of the success of the composite structure. The hydrophilic loofah microfibers were successfully embedded within the matrix with the help of H-boding and dipole-dipole interactions. It is also evident from the SEM images at larger magnifications (500x) that the PHBV nanofibers were well integrated with the loofah fibers and collagen-chitosan matrix walls through molecular interactions of the polar groups in the structures. Moreover, all the scaffolds produced have interconnected and open pores which is a crucial point for cell accommodation.

For hydrogel production, genipin was used as the natural chemical cross-linker for both collagen and chitosan. The covalent crosslinking reaction mechanism between genipin and collagen-chitosan was explained in previous studies [8, 9]. Briefly, genipin can react with the primary amine groups of collagen and chitosan in two different routes. In the first route, SN2 nucleophilic substitution reaction takes place between the ester group of the genipin and the primary amine group of the collagen or chitosan producing a secondary amide bond. In the second route, a tertiary heterocyclic amine linkage is created by an SN1 mechanism through the nucleophilic attack of the primary amine group of the collagen or chitosan to the C3 carbon atom of genipin. FTIR spectra of the composite hydrogel scaffolds are given in Fig. 2b. Characteristic chitosan and collagen peaks and their corresponding chemical groups in the scaffolds appeared at; ~3300 cm^− 1 (O-H and N-H stretching), ~ 2950 cm^− 1 (C-H stretching), ~ 1645 cm^− 1 (amide I band, C = O stretching), ~ 1550 cm^− 1 (amide II band and amine N-H bending), ~ 1233 cm^− 1 (amide III bands, C-N stretching), ~ 1150 cm^− 1 (C-O-C stretching) and ~ 1055 cm^− 1 (C-O stretching). Upon cross-linking with genipin, the absorbance ratio between the 1645 cm^− 1 band and the 1550 cm^− 1 band increased and also the amide III band at 1233 cm^− 1 shifted to 1279 cm^− 1. Loofah mat contributed to the FTIR spectra with the characteristic cellulose peaks of O-H, C-H, and C-O which appeared at similar frequencies with that of collagen and chitosan. Also, the C = O stretching vibration of the ester group at 1721 cm^− 1 indicated the presence of PHBV nanofibers in the structure.

The crosslinking reaction of chitosan and collagen with genipin which was used at three different concentrations allowed the hydrogel properties of the scaffolds to be tuned. In terms of swelling behavior, the uncrosslinked scaffold (PL/ChtCol-0) showed 694 ± 73%, and crosslinked hydrogel scaffolds showed 858 ± 94%, 974 ± 47 and 960 ± 93% swelling ratios with increasing crosslinker ratios, respectively (Fig. 2c). Contrary to expectations, it is seen that the swelling ratio increases slightly as the genipin ratio increases. Similar results were also obtained in the literature [8, 9, 20, 21]. Even though, this increase was found to be statistically insignificant, this might be due to the hydrophilic anisotropic structure of the natural loofah mat and/or molecular interactions of the genipin molecules with the polar groups of collagen, chitosan and loofah. In addition, the composite hydrogel scaffolds' water content values were calculated as 86–92%.

The stiffness and damping properties of the composite hydrogel scaffolds were evaluated by DMA analysis. The variation of the storage and loss modulus and tan δ values of the crosslinked scaffolds as a function of frequency are given in Fig. 3a-b. In all the scaffolds, higher storage modulus values were observed compared to the loss modulus values, as an indication of elastic solid-like behavior. PL/ChtCol-5 scaffold with the highest crosslinker concentration exhibited a sharp decrease in the E' values at lower frequencies, indicating damage due to its more brittle structure. On the other hand, even though PL/ChtCol-1 and PL/ChtCol-3 scaffolds were damaged at similar frequencies, the increase in the E' values in PL/ChtCol-3 scaffold was more pronounced meaning that it had more capacity to store energy. Also, as expected, a slight increase in the storage modulus values of the scaffolds was observed with increasing crosslinker concentration.

The compressive strengths of the composite hydrogel scaffolds are given in Fig. 3c. The PL/ChtCol-3 scaffold showed higher compression strength than the other two scaffolds. Consequently, when all the scaffold characterization results were evaluated, it was decided to perform in vitro and in vivo studies on the PL/ChtCol-3 scaffold.

3.2. In vitro Analysis

In the characterization of stem cell lines, rMSCs were successfully differentiated into chondrogenic, osteogenic and adipogenic cell types. In chondrogenic differentiation, proteoglycans produced in the cell cytoplasm were stained dense blue. The mineralization spots produced by osteoblastic activity were stained red in osteogenic differentiation. Finally, oil droplets that indicate adipogenic differentiation were stained red (Fig. 4a).

3.2.1. In-Vitro Cytotoxicity and Cell Proliferation Analysis

The potential cytotoxic effect of the scaffolds on rMSCs was investigated by LDH activity. The results showed that the level of LDH released from the cells was higher on the 3rd day compared to the 7, 10, and 14 days (Fig. 4b). This statistically significant difference might be caused by the low toxicity of genipin [8]. Due to the decrease in the LDH levels, it is assumed that the scaffolds were non-cytotoxic.

rMSCs’ proliferation on the scaffolds showed an increasing trend during the culture period (Fig. 4c). The cell proliferation showed a statistically significant increase on the 10th and 14th days compared to the 3rd day of the culture period, and this increase was stabilized after the 10th day.

3.2.2. Cell Attachment Studies

The cell attachment was first studied by using SEM Fig. 5a. The cells were indicated by red arrows. It can be seen from days 14 and 21 that rMSCs were well attached and spread on loofah micro- and PHBV nano-fibers. As a result, the structure of the scaffold was found to be suitable for cell proliferation.

In addition, fluorescence microscopy was used to evaluate cell attachment on the scaffolds on days 14 and 21 (Fig. 5b). In the fluorescence images, the nuclei of rMSCs stained with DAPI were blue, whereas the cytoskeleton of rMSCs stained with Actin was red. The images depicted that cells were homogeneously distributed on the surface.

3.2.3. In Vitro Histological and Immunohistochemical Analysis

Sections obtained from the scaffolds in which rMSCs were cultured, were stained histologically (H&E and Masson Trichrome) (Fig. 5c-i and c-ii), respectively and immunohistochemically (Type I and Type II collagen) (Fig. 5c-iii and c-iv), respectively. It was observed that the cells adhered and proliferated well on the scaffolds, especially on day 21. Most of the cells on the surface were round-shaped but there were also fewer elongated cells. In addition, cells preferred to accumulate around the loofahs (Fig. 5d-i and d-ii), respectively). In immunohistochemical stainings, it was observed that collagen type II positivity was more intense on day 21 of incubation (Fig. 5d-iii and d-iv), respectively.

3.3. In-vivo Meniscus Regeneration Analysis

For in-vivo studies, a surgical procedure was applied to the right knee joints of 24 adult male New Zealand Rabbits with normal activity and 2.5-3 kg body weight (Fig. 6a-d). The animals were followed up for 12 weeks. After the 2nd week of the post-operation, the rabbits were fully mobilized and had a full range of motion, the wounds were healed, and shaved hairs began to grow. After the 4th week, pubescence occurred again in the wound areas. The mobility of the rabbits were normal, and no disruption in the nutrition and daily activities of the animals were observed during 12 weeks. There were no findings suggestive of local or systemic infection, and the healing of the wound site was within the expected range. After the healing period, the animals were sacrificed by administering a high-dose anesthetic and the tissues were dissected carefully for further analyses.

3.3.1. Post-implantation Micro-CT Analysis

For Micro-CT imaging, animals were dissected by preserving the knee joints and the tissues were frozen in PBS [22]. Micro-CT images of the cell-laden (a) and cell-free (b) PL/ChtCol-3 scaffolds after the 12-week healing period are given in Fig. 6f-i. As a result of the preliminary evaluations, it was observed that the cell-laden PL/ChtCol-3 scaffold showed a more effective recovery.

In order to evaluate the meniscus tissue with Micro-CT, the tibial plateau of the articular cartilage, which is below the medial meniscus anterior where the scaffolds were placed, was evaluated. Analyzes were performed using DICOM data. In the thin section analysis of Micro-CT, it was seen that the healing process of cartilage damage continued for the cell-free PL/ChtCol-3 scaffold (Fig. 6h). On the other hand, the healing process seemed to be accelerated in the cell-laden PL/ChtCol-3 scaffold (Fig. 6i).

In 3D Micro-CT examinations, no abnormal findings were found on the joint surface where the cell-laden PL/ChtCol-3 scaffold was placed. Similarly, on the joint surface where the cell-free scaffold PL/ChtCol-3 scaffold was located, no lesion formation was observed in the bone tissue. (Fig. 6f-g).

3.3.2. Post-implantation Biomechanical Studies

The biomechanical properties of the tissues after implantation were examined by the compression test. The force-displacement curves obtained were converted to stress-strain curves (Fig. 6e), and elastic modulus values were calculated (Table 2). The compressive modulus values of the meniscus vary between 0.5–1.5 MPa in sections taken from the circumferential, radial, or vertical plane due to its anisotropic structure [23]. When the elastic modulus values were compared, it was seen that the modulus values obtained for all 3 groups were consistent with the compressive modulus of the meniscus. It was determined that the group implanted with the cell-laden PL/ChtCol-3 scaffold, in which the best healing was seen in the Micro-CT analyses, had the highest modulus value, as expected.

Table 2

The average maximum stress, strain, and elastic modulus values were obtained from the compression test performed after the 12-week healing period
	Max Strain (a.u.)	Max Stress (MPa)	Elastic Modulus (MPa)
Empty defect	2.75	1.59	0.58
PL/ChtCol-3 scaffold without rMSCs	1.50	1.28	0.85
PL/ChtCol-3 scaffold with rMSCs	2.25	3.28	1.46

From the compression test results, a statistically significant difference was found between the empty defect and cell-laden scaffold, and cell-laden and cell-free scaffold groups (p < 0.0001). In addition, p = 0.0014 was found when the empty defect and cell-free scaffold groups were compared.

3.3.3. Post-implantation Histological and Immunohistochemical Analysis

H&E staining was applied to the tissues for histomorphological examination. When the groups were compared, it was observed that the volume of the meniscus tissue decreased in the empty defect group compared to the other groups, and in some regions, there were multinuclear (MNL) cell infiltrations. In the cell-laden PL/ChtCol-3 scaffold, it was determined that the cells were distributed within the scaffold and the wound volume decreased compared to the cell-free group by adapting to the damaged tissue. In addition, the cell-laden scaffold appeared to be well connected with the damaged surrounding tissue. It can be stated that the cell-laden scaffold was compatible with the meniscus tissue and did not cause an inflammatory response in the damaged area (Fig. 7a1-d1).

In Masson Trichrome staining which was performed to determine the amount of collagen, it was observed that the amount of collagen in the surrounding tissue increased in the cell-laden scaffold compared to the cell-free scaffold (Fig. 7a2-d2).

Alcian Blue staining was performed to detect the accumulation of GAGs in the extracellular matrix of the cartilage tissue. According to the data obtained, it was observed that the accumulation of GAGs in the cell-laden group was higher than in the cell-free and empty defect group (Fig. 7a3-d3).

Alizarin Red staining was performed to observe the amount of calcification in the cells. It was observed that the calcium deposition increased in and around the scaffold in cell-laden and cell-free groups, and this increase was more in the cell-free group (Fig. 7a4-d4).

Type I and II collagen stainings were performed for immunohistochemical analysis. When the results of type I (Fig. 7a5-d5) and type II (Fig. 7a6-d6) collagen analyses were examined, it can be seen that both cell-free and cell-laden scaffold implanted groups showed more collagen positivity (Fig. 7c5-d5 and 7 c6-d6).

In this study, a novel composite hydrogel scaffold was designed comprising collagen and chitosan polymers with loofah micro- and PHBV nano-fibers. The produced scaffolds showed an interconnected porous structure with a high swelling ratio. PL/ChtCol-3 scaffold which had the best damping capacity and compressive strength was chosen for further in-vitro and in-vivo analysis. Based on the in vitro analyses performed using a commercially obtained rMSCs cell line, PL/ChtCol-3 scaffold was shown to be non-toxic and biocompatible. Moreover, PL/ChtCol-3 scaffold allowed rMSCs to attach and spread homogeneously and it triggered collagen type I and especially type II positivity. In-vivo implantation studies were evaluated by post-implantation Micro-CT, biomechanical, histological and immunohistochemical analyses. Micro-CT and biomechanical analyses indicated a better healing capacity for the cell-laden PL/ChtCol-3 scaffold group with the highest wound volume reduction and compressive modulus value comparable to that of the meniscus. When the results of the H&E, Masson Trichrome, and Alcian Blue stainings were evaluated, it was seen that the cell-laden PL/ChtCol-3 scaffold was more effective in meniscus repair with higher accumulation of GAG and calcium depositions. Also, type I and type II collagen staining results showed that both cell-laden and cell-free scaffolds supported collagen formation. As a result, it was shown that the produced PL/ChtCol-3 scaffold, especially when implanted with rMSCs, is more biocompatible and suitable for meniscus regeneration.

Conflicts of interest/Competing interests

The authors declare that there is no conflict of interest

Ethics Approval
In vivo experimentation was performed with the approval of the Dokuz Eylul University Experimental Animals Ethical Council (Protocol No: 77/2016).

Funding Statement

This study was supported by the Scientific and Technological Research Council of Turkey (TUBITAK) with project number 117M301.

Author Contribution

The authors confirm contribution to the paper as follows: Study conception and design: Hasan Havitcioglu, Aylin Ziylan Albayrak and Gizem Baysan Data collection for scaffold fabrication: Gizem Baysan, Oylum Colpankan Gunes and Aylin Ziylan Albayrak Data collection for in vitro analysis: Gizem Baysan, R. Bugra Husemoglu, Merve Perpelek and Aylin Kara Ozenler Data collection for in vivo studies: Gizem Baysan and Efe Kemal Akdogan Data collection for histological stainings and immunohistochemical analysis: Pinar Akokay and Bekir Ugur ErgurAnalysis and interpretation of results: Hasan Havitcioglu, Aylin Ziylan Albayrak, Efe Kemal Akdogan and Gizem Baysan Draft manuscript preparation: Aylin Ziylan Albayrak and Gizem Baysan All authors reviewed the results and approved the final version of the manuscript.

Acknowledgement

The authors (Gizem Baysan, R. Bugra Husemoglu, Aylin Kara Ozenler and Merve Perpelek) were scholars in the Council of Higher Education (YOK) 100/2000 Doctoral Program in the field of Biomaterials and Tissue Engineering. The authors also thank Izmir Biomedicine and Genome Center (IBG) for the fluorescence microscopy imaging and Ege University, Central Research Test and Analysis Laboratory Application and Research Center (Ege-MATAL) for the Micro-CT analysis.

Data availability

The raw/processed data required to reproduce these findings cannot be shared at this time due to technical or time limitations.

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In Vitro and In Vivo Evaluations of Loofah Micro- and PHBV Nano-Fiber Integrated Hydrogel Scaffolds for Meniscus Regeneration

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials and Methods

2.1. Materials

2.2. Production of PHBV Nanofiber and Loofah Mat Reinforced Collagen-Chitosan Scaffolds

2.2.1. Preparation of Loofah Mats

2.2.2. Preparation of PHBV Nanofibers

2.2.3. Preparation of Composite Sponges and Hydrogel Scaffolds

2.3. Characterization of the Composite Hydrogel Scaffolds

2.3.1. Morphological Characterization

2.3.2. Chemical Characterization

2.3.3. Swelling Test

2.3.4. Viscoelastic Property Characterization

2.3.5. Mechanical Test

2.4. In vitro Studies

2.4.1. Differentiation capacity of rMSCs

2.4.2. Cytotoxicity analysis

2.4.3. Cell proliferation of scaffolds

2.4.4. Cell attachment on scaffolds

2.5. In vivo Studies

2.6. Micro-CT Characterizations

2.7. Post-Implantation Biomechanical Studies

2.8. Histological Stainings and Immunohistochemical Analysis

2.9. Statistical Analysis

3. Results and Discussion

3.1. Production and Characterization of the PL/ChtCol Scaffolds

3.2. In vitro Analysis

3.2.1. In-Vitro Cytotoxicity and Cell Proliferation Analysis

3.2.2. Cell Attachment Studies

3.2.3. In Vitro Histological and Immunohistochemical Analysis

3.3. In-vivo Meniscus Regeneration Analysis

3.3.1. Post-implantation Micro-CT Analysis

3.3.2. Post-implantation Biomechanical Studies

3.3.3. Post-implantation Histological and Immunohistochemical Analysis

4. Conclusion

Declarations

Conflicts of interest/Competing interests

Ethics Approval In vivo experimentation was performed with the approval of the Dokuz Eylul University Experimental Animals Ethical Council (Protocol No: 77/2016).

Funding Statement

Author Contribution

Acknowledgement

Data availability

References

Additional Declarations

Status:

Version 1

Ethics Approval
In vivo experimentation was performed with the approval of the Dokuz Eylul University Experimental Animals Ethical Council (Protocol No: 77/2016).