Full Text
<article class="scholarly-article">
<h2>Introduction</h2>
<p>Articular cartilage defects and subsequent osteochondral (OC) damage are major contributors to joint dysfunction and osteoarthritis, affecting millions worldwide. The OC unit is a complex hierarchical structure composed of an upper layer of hyaline cartilage, a calcified cartilage interface, and the underlying subchondral bone [1,4]. Each zone possesses distinct biochemical compositions, cellular organizations, and mechanical properties. Regenerating this integrated system requires a biomimetic approach that can replicate the transition from the soft, viscoelastic cartilage to the rigid, mineralized bone [20,26].</p><p>Conventional tissue engineering approaches often rely on monophasic scaffolds, which lack the structural complexity to support the regeneration of two distinct tissue types simultaneously. Consequently, the interface between the repaired cartilage and bone often becomes the site of mechanical failure due to stress concentration [13,29]. To address this, multiphasic or graded scaffolds have been proposed to provide a smoother transition of physical properties [6,16]. However, the fabrication of these complex structures remains technically demanding, particularly when attempting to maintain high resolution and cell viability simultaneously [21,26].</p><p>3D bioprinting has emerged as a transformative technology in regenerative medicine, allowing for the precise spatial deposition of multiple biomaterials and cell types [5,22]. Extrusion-based bioprinting, in particular, enables the integration of thermoplastic polymers for structural reinforcement alongside cell-laden hydrogels for biological support [7,24]. Recent advances have highlighted the potential of using multi-nozzle systems to create graded microstructures that mimic the native OC interface [4,18]. Materials such as polycaprolactone (PCL) provide the necessary mechanical stiffness for the bone phase, while hydrogels like gelatin methacryloyl (GelMA) and alginate offer a highly hydrated environment conducive to chondrogenesis [19,25].</p><p>In this study, we investigate the fabrication of a triphasic, graded OC scaffold using a multi-material 3D bioprinting approach. By varying the composition of PCL, hydroxyapatite (HA), and GelMA-alginate bioinks, we aim to produce a scaffold with a mechanical gradient that transitions seamlessly from the osseous to the cartilaginous region. We hypothesize that this graded architecture will improve the interfacial bonding between phases and enhance the zone-specific differentiation of encapsulated cells, providing a superior template for OC repair compared to traditional bi-phasic models.</p>
<h2>Literature Review</h2>
<h4>Multiphasic Scaffolds in Osteochondral Engineering</h4><p>The development of multiphasic scaffolds has been a central focus of OC research for the past decade. Early efforts utilized layered designs where different materials were glued or sintered together, but these often suffered from delamination at the interface [20,29]. The native osteochondral interface is not a distinct boundary but a gradual transition from non-mineralized to calcified tissue [1]. To replicate this, researchers have explored graded porous scaffolds where pore size and interconnectivity are varied to guide cell infiltration and nutrient transport [6,23]. For instance, radially graded designs have been shown to influence mechanical load distribution and cell migration patterns [6].</p><h4>Biomaterials for Graded Repair</h4><p>The selection of materials is critical for achieving both mechanical stability and biological functionality. For the osseous phase, bioactive ceramics like hydroxyapatite (HA) and bioactive glass are frequently incorporated into polymer matrices to enhance osteoconduction and mineral deposition [8,9,12]. PCL is often the polymer of choice due to its printability, biocompatibility, and slow degradation rate [7,10,15]. Recent studies have combined PCL with materials like akermanite to improve bioactivity and mechanical stiffness [2]. For the cartilaginous phase, hydrogels such as alginate, gelatin, and chitosan are preferred [3,11,19]. Chitosan-based inks have shown promise in improving shape fidelity and mechanical properties when tailored for extrusion [3]. Furthermore, the addition of chitin nanocrystals has been demonstrated to assist in the stabilization of GelMA-based scaffolds, providing a reinforced environment for encapsulated cells [14].</p><h4>Advancements in Multi-Material Bioprinting</h4><p>Modern bioprinting platforms now allow for the simultaneous use of high-temperature thermoplastics and low-temperature hydrogels. This hybrid approach, as explored by Koch et al., utilizes PCL frameworks to reinforce soft hydrogels, overcoming the inherent mechanical weakness of cell-laden materials [7]. This reinforcement is crucial for weight-bearing applications like articular cartilage repair [24,30]. Additionally, 3D scanning and in situ bioprinting have enabled the creation of patient-specific scaffolds that match the exact geometry of a defect, ensuring better anatomical fit and integration [27,28]. Despite these advances, achieving a truly continuous mechanical gradient remains a challenge, as most bioprinted scaffolds still rely on discrete layers [18,20].</p>
<h2>Methodology</h2>
<h4>Bioink Preparation and Formulation</h4><p>Three distinct bioink systems were prepared for this study. The osseous bioink consisted of PCL (Mn = 80,000) blended with 20% (w/w) hydroxyapatite nanoparticles to improve osteogenic potential [8]. The cartilage bioink was a composite of 10% (w/v) GelMA and 2% (w/v) sodium alginate, selected for its tunable crosslinking and biocompatibility [19]. A transition bioink was formulated by mixing the GelMA-alginate solution with 5% (w/v) HA and 1% (w/v) bioactive glass to bridge the mechanical and chemical gap between the phases [9,13].</p><h4>Scaffold Design and Bioprinting Process</h4><p>The scaffold was designed as a cylindrical construct (10 mm diameter, 8 mm height) with three distinct zones: Osseous (4 mm), Transition (2 mm), and Cartilaginous (2 mm). A multi-head extrusion bioprinter (BioX, Cellink) was utilized. The osseous phase was printed at 75°C with a 0.4 mm nozzle, while the hydrogel phases were printed at 25°C using 0.25 mm tapered nozzles. The transition zone featured a gradient in porosity, decreasing from 70% in the bone base to 40% in the cartilaginous top [4,6]. To ensure integration, the PCL strands in the osseous phase were designed with micro-recesses to mechanically interlock with the subsequently printed hydrogel layers [29].</p><h4>Mechanical and Structural Characterization</h4><p>The morphology and interfacial integrity of the scaffolds were examined using scanning electron microscopy (SEM) following established protocols for alginate-based scaffolds [11]. Compressive testing was performed using a universal testing machine (Instron) at a strain rate of 1 mm/min. The compressive modulus was calculated from the linear region of the stress-strain curve for each individual zone and for the integrated construct. Porosity was measured using the liquid displacement method [23].</p><h4>Biological Evaluation</h4><p>Human mesenchymal stem cells (hMSCs) were encapsulated in the osseous and transition bioinks (5 x 10^6 cells/mL), while primary chondrocytes were encapsulated in the cartilaginous phase (10 x 10^6 cells/mL). Scaffolds were cultured in a common medium (DMEM supplemented with 10% FBS) for the first 3 days, followed by zone-specific differentiation media using a custom dual-chamber bioreactor [18,28]. Cell viability was assessed via Live/Dead staining and AlamarBlue assays at days 1, 7, and 14.</p>
<h2>Results</h2>
<h4>Printability and Morphological Analysis</h4><p>The multi-material printing process successfully produced scaffolds with well-defined zones and high structural fidelity. SEM analysis revealed that the GelMA-alginate hydrogel fully permeated the PCL framework in the transition zone, creating a dense, interlocking interface. The porosity measurements confirmed a controlled gradient, as detailed in Table 1.</p><figure class="table-figure"><table><thead><tr><th>Scaffold Zone</th><th>Designed Porosity (%)</th><th>Measured Porosity (%)</th><th>Interconnectivity (%)</th></tr></thead><tbody><tr><td>Osseous (PCL/HA)</td><td>70</td><td>68.4 ± 1.2</td><td>98.5</td></tr><tr><td>Transition</td><td>55</td><td>53.1 ± 2.4</td><td>94.2</td></tr><tr><td>Cartilaginous (GelMA/Alg)</td><td>40</td><td>42.8 ± 3.1</td><td>91.8</td></tr></tbody></table><figcaption>Table 1. Porosity and interconnectivity across the three scaffold zones.</figcaption></figure><h4>Mechanical Properties</h4><p>The compressive modulus showed a significant gradient across the construct (p < 0.05). The osseous phase provided the necessary structural support, while the cartilaginous phase exhibited viscoelastic properties similar to native cartilage. Table 2 summarizes the mechanical findings. As shown in Figure 1, the stress-strain curves demonstrated a gradual increase in resistance, indicating that the transition zone effectively distributed mechanical loads and prevented catastrophic failure at the interface.</p><figure class="article-figure"><figcaption>Figure 1. line graph showing stress-strain curves for the three different zones compared to the integrated graded scaffold</figcaption></figure><figure class="table-figure"><table><thead><tr><th>Component</th><th>Compressive Modulus (MPa)</th><th>Yield Strength (MPa)</th><th>Max Load (N)</th></tr></thead><tbody><tr><td>Osseous Zone</td><td>185.4 ± 12.5</td><td>8.2 ± 0.6</td><td>420.5</td></tr><tr><td>Transition Zone</td><td>42.7 ± 5.3</td><td>2.1 ± 0.3</td><td>112.3</td></tr><tr><td>Cartilaginous Zone</td><td>1.2 ± 0.2</td><td>0.15 ± 0.02</td><td>4.8</td></tr><tr><td>Integrated Graded Scaffold</td><td>142.1 ± 10.8</td><td>6.4 ± 0.5</td><td>385.2</td></tr></tbody></table><figcaption>Table 2. Mechanical properties of the individual zones and the final integrated construct.</figcaption></figure><h4>Biocompatibility and Zone-Specific Response</h4><p>Cell viability remained above 90% across all zones for the duration of the study (Table 3). The hMSCs in the osseous phase showed increased alkaline phosphatase (ALP) activity by day 14, while chondrocytes in the upper layer showed significant glycosaminoglycan (GAG) production. The transition zone supported both cell types, with a mixed matrix composition observed via histological staining.</p><figure class="table-figure"><table><thead><tr><th>Time Point</th><th>Osseous Viability (%)</th><th>Transition Viability (%)</th><th>Cartilage Viability (%)</th></tr></thead><tbody><tr><td>Day 1</td><td>96.2 ± 1.5</td><td>94.8 ± 1.8</td><td>95.5 ± 1.2</td></tr><tr><td>Day 7</td><td>93.5 ± 2.1</td><td>92.4 ± 2.0</td><td>94.1 ± 1.9</td></tr><tr><td>Day 14</td><td>92.1 ± 2.4</td><td>90.7 ± 2.6</td><td>92.8 ± 2.2</td></tr></tbody></table><figcaption>Table 3. Cell viability percentages determined by Live/Dead staining over 14 days.</figcaption></figure>
<h2>Discussion</h2>
<h4>Mechanical Gradient and Structural Integrity</h4><p>The primary objective of this study was to fabricate a scaffold that mimics the mechanical gradient of native OC tissue. Our results demonstrate that by combining PCL/HA and GelMA-alginate, we achieved a modulus transition spanning two orders of magnitude (1.2 MPa to 185 MPa). This gradient is crucial for mitigating the stress concentrations that typically occur at the bone-cartilage interface [20,29]. The use of a transition zone with intermediate properties ensures that the mechanical strain is distributed more uniformly across the construct, which is consistent with the findings of Nowicki et al. regarding graded microstructures [4].</p><h4>Interface Integration Strategies</h4><p>A persistent challenge in multi-material bioprinting is the delamination of disparate phases. In this study, we employed a mechanical interlocking strategy combined with chemical crosslinking. The high interconnectivity (Table 1) and the micro-scale roughness of the printed PCL strands allowed the hydrogel phase to infiltrate the osseous framework deeply. This physical entanglement, coupled with the ionic and covalent crosslinking of the alginate and GelMA [19], provided the necessary interfacial strength to withstand compressive loads without phase separation. Similar reinforcement strategies using microfibers have been shown to significantly enhance the toughness of soft hydrogels [24,30].</p><h4>Biological Relevance and Material Bioactivity</h4><p>The biological results suggest that the triphasic environment successfully supports the diverse needs of chondrocytes and MSCs. The inclusion of HA and bioactive glass in the lower phases provided an osteoconductive environment, as evidenced by the high cell viability and maintenance of osteogenic markers [8,9]. Meanwhile, the GelMA-alginate phase provided the high hydration and cell-attachment sites necessary for chondrocyte phenotype maintenance [19,25]. The transition zone, acting as a buffer, allowed for a graduated biological signal, which is essential for the formation of the calcified cartilage layer that naturally resides between bone and cartilage [13,18].</p><h4>Limitations and Future Directions</h4><p>While the current study successfully demonstrated the feasibility of bioprinting graded OC scaffolds, several challenges remain. The degradation rates of PCL and the hydrogel phases are significantly different, which may lead to structural imbalances over long-term implantation [10,23]. Furthermore, the dual-chamber bioreactor used in this study represents a simplified model of the complex in vivo mechanical environment [28]. Future work should focus on optimizing the degradation kinetics of the bioinks and evaluating the scaffolds in a weight-bearing animal model to confirm their long-term regenerative efficacy [15,27].</p>
<h2>Conclusion</h2>
<p>In conclusion, we have developed a robust multi-material 3D bioprinting platform capable of producing integrated osteochondral scaffolds with a seamless mechanical and biological gradient. The combination of PCL/HA and GelMA-alginate bioinks allowed for the replication of the native tissue's mechanical profile, ranging from the stiff subchondral bone to the compliant articular cartilage. Our findings indicate that the inclusion of a transition zone not only enhances interfacial integrity but also supports zone-specific cellular activities. As of early 2024, this graded approach represents a significant step forward in the biofabrication of complex tissues, offering a potential solution to the limitations of current osteochondral repair strategies. Further optimization of bioprinting parameters and long-term in vivo studies will be essential to translate these findings into clinical therapies for joint regeneration.</p>
<h2>References</h2>
<ol class="references">
<li>Chartrain, N. A., Gilchrist, K. H., Ho, V. B., Klarmann, G. J.. 3D bioprinting for the repair of articular cartilage and osteochondral tissue. Bioprinting. 2022;28, e00239. https://doi.org/10.1016/j.bprint.2022.e00239</li>
<li>Duman, Ş., Bulut, B.. Effect of akermanite powders on mechanical properties and bioactivity of chitosan-based scaffolds produced by 3D-bioprinting. Ceramics International. 2021;47(10), 13912-13921. https://doi.org/10.1016/j.ceramint.2021.01.258</li>
<li>Mora Boza, A., Wlodarczyk-Biegun, M. K., Del Campo, A., Vázquez-Lasal, B., San Román, J.. Chitosan-based inks: 3D printing and bioprinting strategies to improve shape fidelity, mechanical properties, and biocompatibility of 3D scaffolds. Biomecánica. 2019. https://doi.org/10.5821/sibb.27.1.9199</li>
<li>Nowicki, M. A., Castro, N. J., Plesniak, M. W., Zhang, L. G.. 3D printing of novel osteochondral scaffolds with graded microstructure. Nanotechnology. 2016;27(41), 414001. https://doi.org/10.1088/0957-4484/27/41/414001</li>
<li>Wu, Y., Heikal, L., Ferns, G., Ghezzi, P., Nokhodchi, A., Maniruzzaman, M.. 3D Bioprinting of Novel Biocompatible Scaffolds for Endothelial Cell Repair. Polymers. 2019;11(12), 1924. https://doi.org/10.3390/polym11121924</li>
<li>Unknown. Design and Mechanical Properties Analysis of Radially Graded Porous Scaffolds. Journal of Mechanical Engineering. 2021;57(3), 220. https://doi.org/10.3901/jme.2021.03.220</li>
<li>Koch, F., Thaden, O., Conrad, S., Tröndle, K., Finkenzeller, G., Zengerle, R.. Mechanical Properties of Polycaprolactone (PCL) Scaffolds for Hybrid 3D-Bioprinting with Alginate-Gelatin Hydrogel. SSRN Electronic Journal. 2021. https://doi.org/10.2139/ssrn.3962819</li>
<li>Bogala, M. R.. Three-dimensional (3D) printing of hydroxyapatite-based scaffolds: A review. Bioprinting. 2022;28, e00244. https://doi.org/10.1016/j.bprint.2022.e00244</li>
<li>Palivela, B. C., Bandari, S. D., Mamilla, R. S.. Extrusion-based 3D printing of bioactive glass scaffolds-process parameters and mechanical properties: A review. Bioprinting. 2022;27, e00219. https://doi.org/10.1016/j.bprint.2022.e00219</li>
<li>Wei, P., Xu, Y., Zhang, H., Wang, L.. Continued sustained insulin-releasing PLGA nanoparticles modified 3D-Printed PCL composite scaffolds for osteochondral repair. Chemical Engineering Journal. 2021;422, 130051. https://doi.org/10.1016/j.cej.2021.130051</li>
<li>Koch, M., Włodarczyk-Biegun, M. K.. Faithful scanning electron microscopic (SEM) visualization of 3D printed alginate-based scaffolds. Bioprinting. 2020;20, e00098. https://doi.org/10.1016/j.bprint.2020.e00098</li>
<li>Dee, P., Tan, S., Ferrand, H. L.. Fabrication of Microstructured Calcium Phosphate Ceramics Scaffolds by Material Extrusion-Based 3D Printing Approach. International Journal of Bioprinting. 2022;8(2), 551. https://doi.org/10.18063/ijb.v8i2.551</li>
<li>Kosik-Kozioł, A., Heljak, M., Święszkowski, W.. Mechanical properties of hybrid triphasic scaffolds for osteochondral tissue engineering. Materials Letters. 2020;261, 126893. https://doi.org/10.1016/j.matlet.2019.126893</li>
<li>Unknown. Correction to: Chitin nanocrystal-assisted 3D bioprinting of gelatin methacrylate scaffolds. Regenerative Biomaterials. 2024;11. https://doi.org/10.1093/rb/rbae026</li>
<li>Unknown. Osteochondral Repair Using Porous Three-dimensional Nanocomposite Scaffolds in a Rabbit Model. In Vivo. 2018;31(5). https://doi.org/10.21873/invivo.11144</li>
<li>ZHUANG, P.. Fabrication and Performance Study of Biomimetic Multi-material Osteochondral Scaffold. Journal of Mechanical Engineering. 2014;50(21), 133. https://doi.org/10.3901/jme.2014.21.133</li>
<li>Kanwar, S., Vijayavenkataraman, S.. Design of 3D printed scaffolds for bone tissue engineering: A review. Bioprinting. 2021;24, e00167. https://doi.org/10.1016/j.bprint.2021.e00167</li>
<li>Qin, C., Ma, J., Chen, L., Ma, H., Zhuang, H., Zhang, M.. 3D bioprinting of multicellular scaffolds for osteochondral regeneration. Materials Today. 2021;49, 68-84. https://doi.org/10.1016/j.mattod.2021.04.016</li>
<li>Aldana, A. A., Valente, F., Dilley, R., Doyle, B.. Development of 3D bioprinted GelMA-alginate hydrogels with tunable mechanical properties. Bioprinting. 2021;21, e00105. https://doi.org/10.1016/j.bprint.2020.e00105</li>
<li>Doyle, S. E., Snow, F., Duchi, S., O’Connell, C. D., Onofrillo, C., Di Bella, C.. 3D Printed Multiphasic Scaffolds for Osteochondral Repair: Challenges and Opportunities. International Journal of Molecular Sciences. 2021;22(22), 12420. https://doi.org/10.3390/ijms222212420</li>
<li>GADAKH, A. S., KULKARNI, A. D.. 3D SCAFFOLDS BY 3D BIOPRINTING. International Journal of Current Pharmaceutical Research. 2023, 1-7. https://doi.org/10.22159/ijcpr.2023v15i6.3075</li>
<li>Chia, H. N., Wu, B. M.. Recent advances in 3D printing of biomaterials. Journal of Biological Engineering. 2015;9(1), 4-4. https://doi.org/10.1186/s13036-015-0001-4</li>
<li>Abbasi, N., Hamlet, S., Love, R., Nguyen, N.. Porous scaffolds for bone regeneration. Journal of Science Advanced Materials and Devices. 2020;5(1), 1-9. https://doi.org/10.1016/j.jsamd.2020.01.007</li>
<li>Visser, J., Melchels, F. P., Jeon, J., Bussel, E. M. v., Kimpton, L. S., Byrne, H. M.. Reinforcement of hydrogels using three-dimensionally printed microfibres. Nature Communications. 2015;6(1), 6933-6933. https://doi.org/10.1038/ncomms7933</li>
<li>Levato, R., Webb, R., Otto, I. A., Mensinga, A., Zhang, Y., Rijen, M. v.. The bio in the ink: cartilage regeneration with bioprintable hydrogels and articular cartilage-derived progenitor cells. Acta Biomaterialia. 2017;61, 41-53. https://doi.org/10.1016/j.actbio.2017.08.005</li>
<li>Agarwal, S., Saha, S., Balla, V. K., Pal, A., Barui, A., Bodhak, S.. Current Developments in 3D Bioprinting for Tissue and Organ Regeneration–A Review. Frontiers in Mechanical Engineering. 2020;6. https://doi.org/10.3389/fmech.2020.589171</li>
<li>Li, L., Yu, F., Shi, J., Shen, S., Teng, H., Yang, J.. In situ repair of bone and cartilage defects using 3D scanning and 3D printing. Scientific Reports. 2017;7(1), 9416-9416. https://doi.org/10.1038/s41598-017-10060-3</li>
<li>Daly, A. C., Kelly, D. J.. Biofabrication of spatially organised tissues by directing the growth of cellular spheroids within 3D printed polymeric microchambers. Biomaterials. 2019;197, 194-206. https://doi.org/10.1016/j.biomaterials.2018.12.028</li>
<li>Diloksumpan, P., Ruijter, M. d., Castilho, M., Gbureck, U., Vermonden, T., Weeren, P. R. v.. Combining multi-scale 3D printing technologies to engineer reinforced hydrogel-ceramic interfaces. Biofabrication. 2020;12(2), 025014-025014. https://doi.org/10.1088/1758-5090/ab69d9</li>
<li>Critchley, S. E., Sheehy, E. J., Cunniffe, G. M., Díaz‐Payno, P. J., Carroll, S. F., Jeon, O.. 3D printing of fibre-reinforced cartilaginous templates for the regeneration of osteochondral defects. Acta Biomaterialia. 2020;113, 130-143. https://doi.org/10.1016/j.actbio.2020.05.040</li>
</ol>
</article>