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Decellularized apple hypanthium as a plant-based biomaterial for cartilage regeneration in vitro: a comparative study of progenitor cell types and environmental conditions

Journal of Biological Engineering volume 19, Article number: 38 (2025) Cite this article

Abstract

Background

Decellularized plant tissues have been shown to enhance the integration and proliferation of human cells, demonstrating biocompatibility. These tissues are now being considered as valuable biomaterials for tissue engineering due to their diverse architectures and favorable cytocompatibility. In this study, we assessed decellularized apple hypanthium as a potential biomaterial for generating cartilage-like structures, utilizing four different progenitor cell types and varying environmental conditions in vitro.

Results

Cell viability assays indicated integration and cell proliferation. Histological staining and gene expression analyses confirmed the synthesis and deposition of a cartilaginous extracellular matrix. Notably, hypoxia had varying effects on chondrogenesis based on the cell type. Among the progenitor cells evaluated, those derived from auricular perichondrium were particularly promising, as they differentiated into chondrocytes without requiring a low-oxygen environment. Additionally, our findings demonstrated that apple-derived biomaterials outperformed microencapsulation in alginate beads in promoting chondrogenesis.

Conclusion

These results highlight the potential of plant-based biomaterials for the development of implantable devices for cartilage regeneration and suggest broader applications in tissue engineering and future clinical endeavors.

Background

The development of innovative three-dimensional (3D) biomaterials for tissue engineering has garnered significant attention over the past decade [1]. These 3D systems exhibit various morphological and biochemical differences compared to traditional 2D cultures on plastic surfaces, making them more representative of the natural extracellular matrix (ECM) environment. They are utilized in a range of applications including drug discovery, cancer cell biology, stem cell research, regenerative medicine and gene expression studies, since they recapitulate cell heterogeneity, structure and functions of primary tissue [2,3,4], Designing effective 3D culture systems necessitates a multidisciplinary approach, incorporating knowledge and expertise in various parameters to select the most suitable materials, cell types, and culture methods for the specific tissue being targeted.

The primary goal of biomaterials is to replicate the proteins and molecules found in the extracellular matrix (ECM) to mimic native tissue and restore physiological functions. Decellularized matrices serve as a promising alternative, as they retain the specific intrinsic signals of native ECM [5]. Consequently, many bioengineering strategies have shifted toward using decellularized matrices [6]. This process involves removing cellular material from tissues and organs, leaving behind the native ECM, which also possesses a non-immunogenic characteristic. These matrices can later be repopulated with the patient’s own cells to create autologous grafts for repairing damaged or injured tissues and organs [7,8,9]. Various animal-derived tissues have been successfully decellularized while preserving their vascular structures, including human skin for soft tissue replacement, retinal matrices for producing thin films [10], as well as constructs for liver [11], cartilage, bone [12, 13] and vascular grafts [14, 15].

In recent years, plants have garnered significant interest due to their various features that resemble mammalian vasculature in terms of morphology, physical properties, and mechanical characteristics. Their availability, biocompatibility, and cost-effectiveness make plant-derived tissues a promising alternative to mammalian and animal tissues and organs [16,17,18]. Notably, decellularized apples have supported the culture of mammalian cells and exhibited high biocompatibility when implanted in vivo in mouse models [19].

Nevertheless, to our knowledge, no study has evaluated the chondrogenesis with plant biomaterial. Therefore, in this study, we tested decellularized apples as a scaffold and evaluated for the first time their potential for promoting chondrogenesis in vitro. We compared various human cell sources for their ability to differentiate into cartilage-like cells, including progenitors from auricular and nasal perichondrium (AuP and NsP), dental pulp stem cells (DPSC), and bone marrow-derived mesenchymal stem cells (BMSC). BMSC are known for their high proliferative capacity and significant chondrogenic potential, making them the gold standard for cartilage repair and regeneration [20, 21]. Recent reports have also investigated the cartilage-forming ability of AuP [22]. These cells can be expanded up to a large number without losing their differentiation potential and have good propensity to produce elastic cartilage [23]. Nasal-septal derived cells are being explored for cartilage engineering, showing promise in repairing osteochondral defects in rat models when encapsulated in type I collagen hydrogel [24]. They have also demonstrated safe and effective regeneration of traumatic knee injuries in clinical trials [25], although research on nasal septum progenitors (NsP) for elastic cartilage regeneration is lacking.

Dental pulp stem cells (DPSC) are also gaining attention for their potential in articular cartilage repair [26]. Characterized by microvilli-like structures that enhance attachment to biomaterials [27], DPSC, when cultured in chondrogenic medium, exhibit rounded morphology associated with increased expression of ACAN and COL2A1 genes in nanocellulose-chitosan hydrogels [28, 29].

Therefore, we compared the four cell types cultured on decellularized apple sections as biomaterials, examining their chondrogenic potential in various culture environments.

Methods

Preparation and decellularization of apple hypanthium tissue

Granny Smith apples (Malus domestica) were purchased from an organic farm and used directly in our laboratory without preservation. The apple hypanthium slices were uniformly cut with a mandolin slicer to achieve sections that were 5 mm thick and 10 mm in diameter. The decellularization was performed by immersion in 1% sodium dodecyl sulfate (SDS, Sigma-Aldrich, Saint-Quentin-Fallavier, France) for 24 h with continuous agitation. After this, the SDS was removed, and the slices were rinsed with distilled water for 30 min. This rinsing step was repeated twice, and the slices were then left in distilled water overnight. Fresh control samples were cut from apples and stored at 4 °C for later use.

DNA extraction, quantification and staining

DNA extraction was carried out following mechanical lysis of the apple tissues with pestle and mortar in lysis buffer consisting of 100 mM Tris-Base, 1% SDS and 10mM dithiothreitol DTT (Sigma Aldrich). After centrifugation to remove debris, isopropanol was added to the supernatant and DNA was precipitated by centrifugation. The pellet was then washed in 70% ethanol before being dried and resuspended in distilled water. The concentration of the DNA was measured by spectrophotometry at 260 nm using micro-drop™ device and SkanIt™ software (Thermo Fisher Scientific, Illkirch-Graffenstaden, France). The values were normalized to the wet weight of the samples, taken directly after slicing. The size of the remaining genomic DNA fragments was assessed by migration on 1% agarose gel (30 min at 100 V) and stained with GelRed stain (Thermo Fisher Scientific). The gel was visualized under UV light and photographed using the ChemiDoc XRS + Gel Imaging System (Bio-Rad, Marnes-la-Coquette, France). Remaining cell nuclei were detected by staining both native and decellularized apple sections with DAPI (Sigma Aldrich).

Cellular sources

Cells were obtained from differents human sources with consent obtained from the patients or their legals, in accordance with local ethics committee guidelines (Comité de Protection des Personnes, CPP Nord-Ouest III, France). Bone marrow-derived mesenchymal stem cells (BMSC) were obtained from bone marrow aspirates of patients undergoing hip arthroplasty. The bone marrow was separated using a Ficoll Paque density gradient (Sigma-Aldrich, France), and the layer of mononuclear cells was collected and seeded in amplification medium consisting of Minimum Essential Medium alpha with glutamine and sodium pyruvate (MEMα, Dutcher, Bernolsheim, France), supplemented with 10% fetal bovine serum (FBS), 0.5 µg/mL basic fibroblast growth factor (FGF-2, Sigma-Aldrich, France), 200 mM glutamine, 100 IU/mL penicillin, and 100 µg/mL streptomycin (Lonza, Levallois Perret, France). The cells were incubated at 37 °C in a 5% CO2 atmosphere, with media changes occurring three times a week, except for the first change after five days of cell adhesion. They were cultured and expanded until the fourth passage before use. At this stage, tests were conducted to confirm the absence of hematopoietic markers (CD34-, CD45-) using RT-PCR.

Human progenitors from nasal (NsP) or auricular (AuP) perichondrium were obtained from surgical waste in the otorhinolaryngology and maxillofacial surgery units of the Caen University Hospital, with consent from the patients or their legal guardians. Following the dissection of the perichondrium (either nasal or auricular), pieces were cut and used for outgrowth cultures. First, small pieces of tissues were put onto plastic wells without medium for 30 min to allow them adhere to plastic. After that, small amount of medium (MEMα with 10% FBS, 1% penicillin/streptomycin, and 0.5 µg/ml bFGF) was added overnight to avoid desiccation. The following day, the whole pieces of tissue were covered by the same medium and this latter was changed once a week, until cells escape from the tissue and grow on the plastic to reach confluency. Cells were then expanded in αMEM culture medium for four passages before being frozen at -150 °C. They were also checked for tri lineage differentiation before future use.

Dental pulp stem cells (DPSCs) were isolated from freshly extracted teeth of young donors (aged 16–20 years), with consent from the patients or their legal guardians. The teeth were first placed in a hypotonic phosphate-buffered saline (PBS) solution, rinsed, and disinfected using an antibiotic solution. The pulp tissue was then carefully extracted from the pulp chamber, minced into small pieces, and subjected to sequential enzymatic digestion using collagenase I (3 mg/ml) and dispase (4 mg/ml, Thermo Fisher) for 30–45 min at 37 °C. After digestion, the resulting cell suspension was centrifuged at 2000 rpm for 10 min, and the cell pellet was resuspended in culture medium (MEMα with 10% FBS, 1% penicillin/streptomycin, and 0.5 µg/ml bFGF). The cells were expanded up to passage 3 and subsequently cryopreserved.

All human cells used here were tested for their ability to tri lineage before use as we already described [23].

GFP-stably transfected rabbit auricular perichondrial cells were previously prepared in the lab and were used here for the viability tests.

Cell seeding and culture

Before use decellularized slices for culturing, they were treated with antibiotics. Briefly, they were immersed for 1.5 h at room temperature with continuous stirring in a solution of 1X PBS and 0.2% penicillin/streptomycin. After that, the samples were incubated in 2 mL of culture medium at 37 °C with 5% CO2 for 12 h in a 24-well plate. The following day, the medium was aspirated, and the samples were left in sterile conditions for 2 h before cell seeding at indicated densities, based on results from preliminary assays. The slices were then placed in 24-well plates and left for 3 h without medium to enhance cell adhesion. Afterward, they were gently covered by medium, and the samples were incubated under standard conditions (37 °C, 5% CO2). After 48 h, they were transferred to new culture plates to avoid the cells that potentially escaped and adhered to plastic. The samples were then either subjected to viability assays or tested for chondrogenesis. This involved incubation under normoxic (21% O2) or hypoxic (3% O2) conditions, with or without chondrogenic medium, for the specified durations, with media changes every two days. Chondrogenic medium composition was: Dulbecco’s Modified Eagle Medium high glucose with glutamine and sodium pyruvate (DMEM, Dutcher), 0.1% antibiotics, 100nM dexamethasone, 50 µg/ml ascorbic acid-2 phosphate, 40 µg/ml proline, 10 ng/ml of Transforming growth factor beta-3 (TGF beta 3) and 1X Insulin Transferrin Selenium media supplement, (ITS + 1). All materials are from Sigma-Aldrich, unless mentioned.

Cell viability

Cell viability was first assessed by measuring Adenosine triphosphate (ATP) using the Cell Titer-Glo® kit (Promega, Charbonnières, France). After cell culture, recellularized slices were lyzed in Cell Titer-Glo Reagent (150 µL) under stirring for 45 min at room temperature, then the emitted luminescence was measured using a luminometer (Varioskan Lux, Thermo Fisher Scientific). This method permits to quantify the ATP present in cell culture, signaling the presence of metabolically active cells. Alternatively, GFP-expressing rabbit AuP were seeded on apple slices and followed by fluorescence observation in EVOS cell imaging system microscope (Thermo Ficher Scientific).

Histology

When needed, the cultured slices were fixed in 4% paraformaldehyde (Sigma-Aldrich) for 1 h at room temperature, then rinsed with 30% sucrose-PBS for 2 h. The slices were cryopreserved in OCT (Optimal Cutting Temperature compound) at -80 °C. Histological sections, 10 to 15 μm thick, were prepared using a Leica CM3050 cryostat (Thermo Fisher Scientific) and mounted on slides. Various stains were applied, including Hematoxylin-Eosin (HE), Safranin-O (SO), Alcian Blue (AB), and Masson’s Trichrome (MT).

RNA extraction and real time RT-PCR

Total RNA was extracted using the Qiagen RNeasy® Mini Kit (Qiagen, Courtaboeuf, Les Ullys, France) following the manufacturer’s protocol. The RNA was treated with DNase (Sigma-Aldrich) and subsequently reverse-transcribed into cDNA using Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase (Invitrogen by Thermo Fisher Scientific) and oligo-dT primers. Amplification was performed using a StepOnePlus™ Real-Time PCR System (Applied Biosystems™ by Thermo Fisher Scientific) with Power SYBR Green mix (Thermo Fisher Scientific). The relative expression of the target gene was calculated and normalized to three housekeeping genes (GAPDH, RPL13, and β2MG). The real-time RT-PCR primers are listed in the Table 1 below:

Table 1 List of the human primers used
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Statistical analyses

Data are presented as mean ± standard deviation of triplicates. Statistical analyses were performed by two-way ANOVA with Tukey’s multiple comparison test, to compare different sets of data, using Graph Pad prism 7 software. Significance level was set at P˂0.005.

Results

Decellularized apple tissue supports human progenitor cells culture

Treatment of apple slices with 1% SDS resulted in a translucent appearance after cell removal (Fig. 1-A). The success of decellularization (Dcl) was confirmed through DAPI staining, which revealed the absence of nuclei (Fig. 1-B), and by quantifying less than 50 ng of DNA remaining per mg of tissue (Fig. 1-C). Additionally, these DNA fragments were smaller than 200 bp (Fig. 1-D), meeting the criteria for effective decellularization.

Fig. 1
figure 1

Decellularization of apple hypanthium tissue. (A): Macroscopic aspect of apple sections before (native) and after decellularization (Dcl). (B): DAPI staining of native and decellularized apple’s sections. Scale bar: 500 μm. (C): Quantification of DNA remaining before and after decellularization (*p˂0.05). (D): Electrophoretic migration of DNA fragments in native and decellularized tissues

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The apple slices were subsequently tested for cellular viability and seeded with GFP-stably transfected perichondrial cells at a density of 2.106 cells per slice. Fluorescent microscopy showed numerous cell clusters with strong green fluorescence (Fig. 2-A). Furthermore, ATP production was measured up to 21 days post-recellularization, indicating good cell viability and even growth after two weeks, in contrast to decellularized control slices, which showed no luminescence (Fig. 2-B). Total DNA measurement in apple disks was performed 7 and 21 days after recellularisation and also suggest an increase in the number of cells (Fig. 2C).

Despite this, for the following experiments, we have opted to increase the cell density at seeding in order to enhance ECM secretion and tissue formation.

Fig. 2
figure 2

Cell viability on decellularized cellulosic matrix of apples. (A): Assessment of GFP expression in stably transfected perichondrial cells after 14 days of culture, visualized in phase contrast, fluorescence and by the superposition of two images. Scale bar: 500 μm. (n = 2). (B): ATP quantification in cells as a function of culture time in recellularized slices compared to a control (decellularized unseeded apple slices) (n = 3). (C) DNA measurement in apple disks 7 and 21 days after cell seeding (*p˂0.05) (n = 3)

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Cellularized apple biomaterials induce the formation of cartilage-like tissues

After optimizing cell seeding into apple biomaterials, we evaluated the chondrogenic potential of different human progenitor cells as described earlier. Cells were seeded at a density of 7.106 cells per decellularized apple slice in DMEM medium and allowed to adhere for 48 h. Following this, they were incubated in either DMEM (Ctrl) or chondrogenic medium (Chondro) under normoxic or hypoxic conditions, except for auricular perichondrium progenitors. For these cells, previous studies demonstrated that hypoxia does not enhance their chondrogenic differentiation, so they were tested only under normoxic conditions [23].

Macroscopic images of the cellularized apple biomaterials were captured after 14 days of culture. Careful observation revealed a whitish, translucent tissue in the center of several samples (Fig. 3). Chondrogenic medium was sufficient to stimulate significant tissue formation in DPSCs and AuP under normoxic condition, while BMSC and NsP required a hypoxic environment to promote visible extracellular matrix (ECM) production.

Fig. 3
figure 3

Macroscopic aspects of apple biomaterials seeded with different progenitors for chondrogenic assay: Apple biomaterials were seeded with bone marrow-derived stem cells (BMSC), nasal perichondrial cells (NsP), dental pulp stem cells (DPSC) and auricular perichondrial cells (AuP), cultured in regular (Ctrl) or chondrogenic (Chondro) media under normoxic (21% O2) or hypoxic (3%O2) atmosphere for 14 days before macroscopic photography

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The cellularized scaffolds were then fixed and processed for histological staining, compared to unseeded decellularized apple slices. Hematoxylin-Eosin (HE), Safranin O (SO), Alcian Blue (AB), and Masson’s Trichrome (MT) stains were used to analyze tissue morphology and ECM composition. Hypoxia significantly enhanced tissue formation in apple scaffolds seeded with BMSCs. This was evidenced by abundant cytoplasmic components and ECM production, including collagen and elastin fibers stained in pink, and cartilage proteoglycans and glycosaminoglycans stained in dark red and blue, respectively, using Safranin O (SO) and Alcian Blue (AB) staining. Collagen fibers were particularly prominent when stained with Masson’s Trichrome (MT), appearing in dark blue and suggesting a high concentration (Fig. 4-A).

In parallel, NsP and DPSC were tested under the same conditions. Both cell types exhibited tissue formation with substantial ECM secretion in response to chondrogenic medium, and hypoxia did not notably enhance this process (Fig. 4-B, C). Similar results were previously observed for AuP [23], the reason why they were tested here only under normoxic conditions, showing abundant tissue formation (Fig. 4-D).

Fig. 4
figure 4

Histological staining. BMSC (4-A), NsP (4-B), DPSC (4-C), AuP (4-D) cells cultured for 14 days under chondrogenic induction in normoxic and hypoxic were stained for histology. HE: Hematoxylin-Eosin, SO: Safranin O, AB: Alcian blue, MT: Masson’s trichrome. Scale bar: 500 μm

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Chondrogenic differentiation of different progenitors within apple biomaterials

The formation of cartilage-like tissue was further confirmed by elevated levels of mRNA associated with chondrogenic differentiation. Cartilaginous genes, including COL2A1, ACAN, COMP, and SOX9, were analyzed by real-time RT-PCR for each cell type. The expression of COL1, which is typically absent in healthy cartilage, was also examined. As expected, BMSCs, used as the gold standard for chondrogenesis, showed upregulation of key chondrogenic genes (COL2, SOX9, ACAN, ELN) when cultured in chondrogenic medium, with further enhancement under hypoxic conditions. However, COL1 was also upregulated, though its expression was reduced under low oxygen exposure (Fig. 5-A).

Nasal progenitors exhibited distinct expression profiles. COL2A1, ELN, and ACAN genes were upregulated by chondrogenic medium but were not significantly affected by hypoxia. In contrast, hypoxia notably increased the expression of SOX9 and COMP (Fig. 5-B). Interestingly, COL1 expression was elevated in both normoxic and hypoxic conditions. In DPSCs, key cartilage markers (COL2A1, ACAN, SOX9) were significantly downregulated, while COL1 was overexpressed (Fig. 5-C).

For AuP cells, which were only tested under normoxic conditions (as hypoxia had previously shown no benefit for their chondrogenesis [23]), the cartilage markers were strongly upregulated, indicating a high chondrogenic potential. Notably, COL1 was downregulated at the same time (Fig. 5-D), highlighting their suitability for proper cartilage formation.

Fig. 5
figure 5

Expression of cartilage markers. BMSC (5-A), NsP (5-B), DPSC (5-C) and AuP (5-D) cells were cultured in apple slices for 14 days in different culture conditions. Relevant genes expression was investigated by real time RT-PCR. ND: normoxia and DMEM (control), NC: normoxia and chondrogenic medium, HD: hypoxia and DMEM (control), HC: hypoxia and chondrogenic medium (n = 3, *p˂0.05, **P˂0.01, ***p˂0.005)

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Cellularized apple biomaterials surpass chondrogenic potential of alginate beads

Decellularized apple emerges as a promising scaffold for promoting chondrogenesis, as it effectively guides various progenitor cells to differentiate into cartilage-like tissues. To assess its chondrogenic potential, we compared it to alginate hydrogel, a well-established and robust in vitro model in this field [30,31,32,33,34,35]. AuP cells were either seeded onto apple scaffolds or encapsulated in alginate beads and cultured in chondrogenic medium for two weeks under normoxic conditions. Gene expression levels were normalized to those observed in the alginate condition.

Interestingly, while ACAN expression was similar in both scaffolds, other key cartilage genes (COL2A1, SOX9, ELN) were significantly upregulated by 2–3 fold in the decellularized apple biomaterials. Notably, apple slices also exhibited a more favorable cartilage profile due to the marked downregulation of COL1 and RUNX2 compared to alginate hydrogel. Expression of the marker of hypertrophy COLX was also slightly decreased but apparently not significantly (Fig. 6). These results suggest a superior potential of decellularized apple scaffolds to drive chondrogenesis at least in vitro.

Fig. 6
figure 6

Comparison of chondrogenesis in apple biomaterials Vs alginate microspheres. Chondrogenic and hypertrophic markers expression in perichondrial cells were compared after seeding into apple biomaterials or alginate beads 14 days of culture in normoxic conditions. mRNA expression of relevant genes was calculated using 3 housekeeping genes and normalized to alginate condition (n = 3, *: p˂0.05, **: P˂0.01, ***: P˂0.001)

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Discussion

Despite significant advancements in tissue engineering, no material has yet been able to fully replicate the complexity of native tissues or restore their optimal function. As a result, the development of novel composite materials that can effectively mimic native tissues remains an ongoing challenge.

Decellularized tissues have emerged as promising alternatives for tissue engineering due to their 3D structure, which provides a scaffold for cells to form tissue-like structures, better mimicking native tissue and enhancing functionality in a more physiologically relevant way. While many successful engineered constructs have utilized decellularized animal tissues [36,37,38], decellularized plant tissues are now being recognized as serious alternatives and could be investigated and used for tissue engineering applications [16, 19, 39, 40]. Numerous studies have shown that they can serve as supportive scaffolds for in vitro 3D culture and can also function as a biocompatible implantable materials in vivo. For example, spinach leaves have been successfully colonized with human endothelial cells and cardiomyocytes derived from stem cells, leading to the development of contractile function [17, 41, 42]. Onion green leaves have emerged as a simple and cost-effective biomaterial for skeletal muscle tissue engineering, where their microstructure has facilitated the differentiation of colorectal adenocarcinoma (Caco-2) cells into aligned myotubes [43]. Plant-derived biomaterials not only provide functional scaffolds but also demonstrate excellent cytocompatibility and biocompatibility due to their low immunogenicity [19, 44, 45]. Moreover, their porous architecture creates a 3D environment that promotes cell growth and tissue formation by facilitating cell infiltration and colonization, while also supporting various cellular biochemical properties including differentiation [40, 43, 45, 46]. In addition, they offer advantages such as lower costs and fewer ethical concerns compared to animal-derived sources [43, 47].

Although further optimization is needed to achieve specific biological and mechanical properties, apple tissue has already shown potential as a natural biomaterial for regenerating adipose, bone, and tendon tissues [18, 48]. To the best of our knowledge, no study has yet investigated the chondrogenic potential of apple tissues and thus, this study is the first to explore apple matrices as biomaterials for auricular cartilage engineering.

Before studying chondrogenic potential of apple-derived matrices, we first decellularized them to remove cellular components and remnants. Decellularization using SDS proved effective and met the standard criteria for complete cell removal [34]. Alternative methods could also be explored, as they may affect differentially the remaining extracellular matrix.

Nevertheless, decellularized apple tissues were cytocompatible and supported cell growth with cells effectively penetrating and integrating into the biomaterial. This aligns with previous studies, where cell proliferation was sustained for up to 10 weeks in apple biomaterials [44].

In addition, concerning more specifically the cell adhesion, cellulose is known to enable cell attachment. Indeed, cells were already described to adhere to the hydrophilic hydroxyl moieties of cellulose and specialized cellulose binding domains [49,50,51]. In another hand, the environment may also play a role in attachment and cell behavior. For example, the nanoscale distribution of adsorbed proteins onto the scaffold, can affect and improve cell adhesion [52].

Hence, the pre-incubation with culture medium prior to seeding in our procedure may induce protein adsorption to the scaffold which promotes cell adhesion.

We focused on chondrogenesis, comparing four types of human progenitor cells derived from nasal and auricular perichondrium, dental pulp, and bone marrow. All cell types exhibited varying degrees of tissue formation when cultured in chondrogenic medium. Cartilage formation in BMSCs requires hypoxia to be enhanced both in vitro and in vivo, consistent with previous findings using these cells [53, 54]. Interestingly, neither NsP nor DPSC were influenced by low oxygen tension, as significant tissue formation was observed under normoxic conditions in both cases. A similar effect was previously observed with AuP cells, which is why they were only tested in normoxia here. This differential response to hypoxia may be related to the differentiation status of the progenitor cells used, potentially due to variations in the repertoire of adhesion molecules depending on cell type. Hypoxia has been reported to modulate the expression of adhesion molecules in MSCs during long-term incubation [53], which can impact cell-cell and cell-scaffold interactions, thereby altering cell behavior and gene expression. It is also possible that during hypoxia, cellular adhesion and mobility change, promoting spatial rearrangement within the scaffold and triggering differential gene expression.

In terms of chondrogenic potential based on gene expression profiles, both NsP and BMSC cells demonstrated the ability to produce cartilage-like tissue, with increased expression of cartilage markers. As noted, hypoxia enhances chondrogenic differentiation in BMSCs but has little effect on NsP cells. However, both cell types still expressed significant levels of COL1, an undesirable protein in cartilage formation. DPSCs, on the other hand, failed to express key cartilaginous genes, and hypoxia appeared to inhibit their differentiation along this lineage. This outcome was unexpected, as previous studies demonstrated the chondrogenic capacity of DPSCs when encapsulated in a 3D alginate model [23]. Numerous studies have documented the ability of DPSC to evolve towards 3 lineages, i.e. chondrogenic, adipogenic, and osteogenic [26, 54]. The overexpression of COL1 observed in DPSC cells likely reflects their tendency to shift towards an osteogenic phenotype, promoting bone tissue formation. The osteogenic potential of DPSCs was explored in bone regeneration, highlighting stem cells from human teeth as an excellent source for bone repair with minimal surgical invasion. Taken altogether, future research should focus on optimizing the osteogenic differentiation of DPSCs on decellularized apple biomaterials to better understand their mechanisms and primarily target bone tissue regeneration.

We have previously demonstrated that AuP cells exhibit strong potential for auricular cartilage regeneration in vitro, and that hypoxia does not enhance their chondrogenesis [23]. In this study, we confirm that AuP cells are the most promising candidates for cartilage engineering among those tested. Under chondrogenic conditions in a normoxic environment, AuP cells significantly upregulated cartilage-specific genes (with only a slight increase in ACAN expression). Notably, COL1 expression was reduced by half, indicating their strong capacity to differentiate into proper cartilage tissue. Histological analysis further supported these findings, showing high levels of proteoglycans, mucins, and cartilage-like tissue, as demonstrated by Alcian Blue and Safranin O staining. Progenitor cells derived from auricular cartilage have demonstrated the capacity to form cartilage and exhibit a chondrogenic profile when cultured in 3D hydrogels [22]. They are also known for their high proliferative rate [55, 56] and multipotent differentiation abilities in several species [57]. Porcine perichondrium-derived progenitors have been shown to generate cartilage-like matrix [58] and this cell type has successfully facilitated the formation of mature, elastic cartilage in vivo in both monkeys and mice [59]. Another advantage of AuP cells in the present work is the relative low expression of COL1, which is not expected in cartilage. Therefore, consistent with previous studies, AuP cells proved to be the best cell source for chondrogenesis when seeded onto apple-derived biomaterials. In addition, they are derived by outgrowth from small, minimally invasive biopsies of auricular perichondrium, making them easy to obtain.

After decellularization, the apple tissue served as a highly suitable scaffold for supporting chondrogenesis, suggesting its potential use in cartilage tissue engineering. Many natural and synthetic scaffolds have been used to promote chondrogenesis in vitro. One of them is microencapsulation of cells in 3D alginate hydrogels in presence of chondrogenic medium [30, 34, 60]. Interestingly, seeding cells onto apple-derived biomaterials resulted in higher expression of cartilage-specific genes compared to 3D alginate. Remarkably, COL1 expression was also reduced, indicating that apple matrices surpass alginate in the support of chondrogenic potential by enhancing desirable cartilage gene expression while limiting unwanted ones. In addition, apple tissue is known to contain compounds such as procyanidins and phloretin that shown to support mitochondrial biogenesis and maintain proteoglycan balance [61] and prevent the degradation of type II collagen and aggrecan in chondrocytes [62]. This is in line with the use of apple tissue and its benefits for cell culture for chondrogenesis purpose.

In order to classify the decellularized apple tissue as a proper scaffold for cartilage engineering, further analyses are still required. Indeed, degradation tests and cartilage-like matrix production in the engineered cartilage should be done to evaluate its durability and composition, respectively, as well as scaffold microstructure by Scanning Electron Microscopy. In addition, biomechanical properties should be measured, particularly in in vivo animal models, to assess its elasticity and strength. Finally, the comparison should be carried out with products used routinely in clinical practice, to assess geometry and mechanical stability of the newly formed tissue. These tests will provide deeper insights and help pave the way for its potential use in clinical trials.

Conclusion

In summary, we showed that decellularized apple tissue can host human progenitor cells proliferation and differentiation. Under appropriate environment, such scaffold is able to drive and improve in vitro chondrogenesis of different cell types. However, progenitors from auricular perichondrium are more likely able to produce cartilaginous tissue when cultured in apple scaffolds.

Data availability

No datasets were generated or analysed during the current study.

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Acknowledgements

The English version of the manuscript was read and corrected by ad hoc staff in the appropriate department of the university of Caen Normandie.

Funding

This work was done in the framework of FHU SURFACE and was partially supported by Fondation des Gueules Cassées, Grant#10-2021). MH and JD were recipients of a PhD and master grants respectively, both from University of Caen Normandy.

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Author notes

  1. Mira Hammad and Justin Dugué contributed equally to this work.

Authors and Affiliations

  1. Laboratoire BioConnect UR 7451, Université de Caen Normandie, Esplanade de la paix CS14032, Caen, 14032 Caen Cedex 5, France

    Mira Hammad, Justin Dugué, Catherine Baugé & Karim Boumédiene#

  2. Fédération Hospitalo Universitaire SURFACE, Amiens, Caen, Rouen, France

    Mira Hammad, Justin Dugué, Catherine Baugé & Karim Boumédiene#

  3. Service ORL et chirurgie Cervico-faciale, CHU de Caen, Caen, France

    Justin Dugué

  4. Phind Inserm UMR-S 1237, Université de Caen Normandie, Caen, France

    Eric Maubert

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  5. Karim Boumédiene#
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Contributions

MH: Investigation and draft preparation, JD: investigation and draft preparation; EM: Investigation/validation; CB: Conceptualization, Reviewing, KB: Conceptualization, validation, writing, draft preparation, supervision. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Karim Boumédiene#.

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Cells were obtained from different human sources during surgical operations such as arthoplasties, rhinoplasties, dental extraction or auricular reconstruction. The consents were obtained from the patients or their legals, in accordance with local ethics committee guidelines (Comité de Protection des Personnes, CPP Nord-Ouest III, France).

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Hammad, M., Dugué, J., Maubert, E. et al. Decellularized apple hypanthium as a plant-based biomaterial for cartilage regeneration in vitro: a comparative study of progenitor cell types and environmental conditions. J Biol Eng 19, 38 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13036-025-00502-2

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Journal of Biological Engineering

ISSN: 1754-1611