3D Niche-Inspired Scaffolds as a Stem Cell Delivery System for the Regeneration of the Osteochondral Interface

Abstract

The regeneration of the osteochondral unit represents a challenge due to the distinct cartilage and bone phases. Current strategies focus on the development of multiphasic scaffolds that recapitulate features of this complex unit and promote the differentiation of implanted bone-marrow derived stem cells (BMSCs). In doing so, challenges remain from the loss of stemness during in vitro expansion of the cells and the low control over stem cell activity at the interface with scaffolds in vitro and in vivo. Here, this work scaffolds inspired by the bone marrow niche that can recapitulate the natural healing process after injury. The construct comprises an internal depot of quiescent BMSCs, mimicking the bone marrow cavity, and an electrospun (ESP) capsule that “activates” the cells to migrate into an outer “differentiation-inducing” 3D printed unit functionalized with TGF-β and BMP-2 peptides. In vitro, niche-inspired scaffolds retained a depot of nonproliferative cells capable of migrating and proliferating through the ESP capsule. Invasion of the 3D printed cavity results in location-specific cell differentiation, mineralization, secretion of alkaline phosphatase (ALP) and glycosaminoglycans (GAGs), and genetic upregulation of collagen II and collagen I. In vivo, niche-inspired scaffolds are biocompatible, promoted tissue formation in rat subcutaneous models, and regeneration of the osteochondral unit in rabbit models.

1. Introduction

Osteochondral defects arise from the progressive degeneration of the articular cartilage reaching the subchondral bone or from injuries due to trauma, leading to reduced mobility and often times to patient disability.^[1] Cartilage of the osteochondral interface accounts for a hierarchical and zonal organization of heterotypic cells (chondrocytes) and associated extracellular matrix, which ultimately defines the outstanding mechanical properties of the tissue. Current clinical treatments are still centred on the natural healing of the tissue by stimulating migration of cells from the bone marrow into the defect, or by implantation of in vitro expanded autologous chondrocytes.^[2] However, this has repeatedly proved unsuccessful in the long-term due to the formation of fibrotic cartilage, hence impairing mechanical and functional properties of the osteochondral unit. The field of tissue regeneration has taken on-board the challenge by investigating multiple solutions that are based on either the implantation of designer cell-free or cell-laden scaffolds with relative success.^[3] Common approaches to scaffold design have converged strategies of cartilage and bone regeneration creating multiphasic scaffolds that mimic key aspects of these tissues such as their biological composition^[3b,d,e,4] and structure,^[3h,5] or include biological factors^[3a,c,6] that, upon release, can promote the specific differentiation of cells and tissue formation. To this end, many studies investigated the potential combination of hydrogel-based scaffolds, aimed at regenerating the cartilage phase, with stiffer—traditionally thermoplastic-based—3D printed materials. The use of mechanically distinct materials stems from the inherent properties of the two tissues, with cartilage having lower mechanical properties than subchondral bone. However, while the mechanical properties of the scaffolds aimed at bone regeneration will be quickly reinforced by the natural mineralization process, hydrogels aimed at supporting cartilage growth appear to be mechanically “too weak”, considering their typically faster degradation rate. Hence, quickly after the formation of neo-tissue the scaffold is cleared from the area and cartilage has to be able to withstand the loads applied into the knee, which are transferred to the cells leading to an “overstress” and long-term failure.^[7] In fact, Mancini et al. recently developed a cell-seeded multiphasic scaffold composed of a 3D printed bone anchoring phase and a fiber-reinforced hydrogel, and showed that regeneration of the osteochondral interface in large (equine) animals at long-term (16 and 24 weeks, compared to the traditional 12 weeks) time points results in macroscopically filled defects that show, however, histological limited functionality.^[7a] Most of current studies result in the formation of fibrocartilagenous tissues, at best, with low glycosaminoglycan content and the production of an extracellular matrix with a higher content of collagen I than the characteristic collagen II.

Cell-free systems rely on the invasion of host bone-marrow derived stem cells (hBMSCs) to infiltrate, populate, and differentiate into the specific phenotypes found in subchondral bone and cartilage. However, these idealized systems are realistically inefficient in vivo due to the scarce population of hBMSCs that represents less than 0.001% of the heterogeneous population of the bone marrow niche.^[8] Other existing tissue-resident cells, such as chondrocytes or osteoblasts, will also infiltrate the damaged area with the inconvenience that the former will dedifferentiate upon proliferation due to an anabolic-catabolic imbalance.^[9] Thus, hBMSC-laden designer scaffolds, where cells are predifferentiated in vitro appear a priori as promising alternatives to regenerate the osteochondral interface. In vitro expansion and re-implantation of autologous BMSCs is approved by the Food and Drug Administration in the USA and the European Commission (1394/2007).^[10] Nevertheless, it is known now that implantation of hBMSCs results in a low cell survival rate^[7a,11] and their expansion in vitro results in loss of stemness, due to a decrease in telomerase activity and alterations of chromosomal morphology associated to senescence and to phenotype changes.^[12] Recently, de Ruijter et al. have convincingly proved in an equine model that the presence and preculture of cells in implanted scaffolds has no significant effect compared to bare scaffolds, highlighting the low cell survival rate reported earlier. Moreover, the study also demonstrates the importance of scaffold structural reinforcement in the cartilage phase to obtain stable defect closure.^[13]

Natural organ regeneration in humans is limited to the embryonic stages. However, tissues—excluding hyaline cartilage— respond to healing by a series of general events that include hematoma, clot formation, and activation of platelets. The latter secretes a series of growth factors and cytokines that recruit immune cells. This inflammatory environment, in return, attracts resident and/or circulating stem cells (amongst other cells) to heal the damaged area.^[14] In an attempt to circumvent the short-comings of the absence of vasculature in cartilage, the scarce population of stem cells in the bone marrow and the early loss of implanted cells in scaffold-based regenerative strategies,^[7a] here we designed bone marrow niche-inspired scaffolds. Niche-inspired scaffolds contain a pool of stem cells with a nonproliferative character that are protected from loss after implantation and would not require an in vitro expansion step. Stem cells in niche-inspired scaffolds can be activated, migrating and proliferating through a secondary electrospun (ESP) compartment, and finally invading a “differentiating” compartment based on peptide-functionalized 3D printed scaffolds. We hypothesized that these bone marrow niche-inspired scaffolds would serve as a depot of cells capable of regenerating the osteochondral interface effectively.

2. Results and Discussion

2.1. Fabrication of Multicompartmental Niche-Inspired Scaffolds

The osteochondral unit comprises the cartilage and subchondral bone interface. Current strategies to regenerate the tissue make use of scaffolds alone or seeded with hBMSCs previously expanded in vitro. In vitro culture presents associated limitations arising from cell isolation, expansion, loss of stemness, uncontrolled differentiation or senescence. In vivo, however, resident stem cells are activated from the nearest source, proliferating, invading, and differentiating to regenerate the damaged tissue. Inspired by the native mechanism of regeneration, we designed niche-inspired scaffolds that serve as a cell-depot and specific tissue formation instructors. Niche-inspired scaffolds were designed as compartmentalized, yet continuous, units that enable the storage of quiescent hBMSCs, their proliferation, and final differentiation into chondrocytes or osteoblasts, reconstructing the osteochondral unit over time.

To do so, we designed a Matryoshka-like niche-inspired scaffold comprised of (i) a hydrogel, aimed at storing quiescent hBM-SCs; (ii) a fibrous cup-shaped ESP mesh containing the hydrogel and aimed at promoting cell proliferation and migration and; (iii) an outer 3D printed biphasic scaffold bearing growth factor-derived peptide sequences aimed at specific cell differentiation— chondrogenic and osteogenic—and tissue formation. The hydrogel was made from 1% w/v alginate functionalized with arginyl-glycyl-aspartic acid (RGD) sequences and accounted for a storage modulus below 5 kPa. We have recently shown that this particular hydrogel is capable of bringing hBMSCs to a nonproliferative and quiescence state where cells enter the G0 phase of their cycle, improving their multilineage differentiation potential after recovering them from the hydrogel.^[15]

The cup-shaped ESP mesh was fabricated from poly(ethylene oxide terephthalate)/poly(butylene terephthalate) (PEOT/PBT) (Figure S1, Supporting Information). The 3D printed biphasic scaffold was fabricated from end-group functionalized polycaprolactone-azide (PCL-azide) and PCL-maleimide, for each of the scaffold phases, with a woodpile pore architecture, 550 μm in strand distance and a fiber diameter of ≈300 μm.

Of outmost importance is the combination of these individual compartments into a continuous niche-inspired scaffold. Four main routes are generally contemplated: mechanical fastening by interlocking features or external devices such as fasteners, direct bonding by nonfusion processes, fusion welding, or with the use of intermediate materials such as glues.^[16] Mechanical fastening approaches require that the joined structures are mechanically robust, which was not applicable to our hydrogel and ESP scaffold compartments. Direct bonding generally requires the use of lasers or other devices that introduce localized heat or promote the roughening of the interfaces that will then remain together. This process, while attractive to our application would require the use of specific instrumentation by the surgeons, and hence was discarded. The use of interfacial materials such as fibrin glue is a common practice in the biomedical field. However, the introduction of an external material could mask the imparted biofunctionality to the fabricated devices and/or cover the predesigned porosity hindering cell migration. Melt welding is an attractive solution for bulk structures, but the lack of control over the integrity of the micrometer features present in our 3D printed (3DP) and ESP devices, together with the possibility of chemical degradation of the introduced biofunctionality, made this technique unrealistic. Thus, we decided to take a dissolution-blending approach for the 3DP and ESP devices by simply dissolving a thin layer of the interface and bringing them into close contact, and a casting approach for the ESP and hydrogel interfaces.

Following the selected dissolution-blending approach, we choose chloroform to bind the 3DP and ESP scaffolds, as this is a common solvent for both polymers and accounts for a low boiling point (≈60 °C) indicative of its high volatility at normal ambient conditions. First, we joined PCL to PCL (3DP to 3DP) and PCL to PEOT/PBT (3DP to ESP) to evaluate the quality of the formed interface (Figure S2, Supporting Information). PCL-PCL 3DP structures joined by dissolution blending showed a thin interface detectable only by a slight misalignment of the two structures. The polymer was only dissolved at the interface, which allowed a good preservation of the printed structure and continuity across the interface. The interface between the 3DP PCL and ESP PEOT/PBT was virtually inexistent, showing a remarkably well-preserved fibrous structure for both devices. This process was initially reported by Camarero-Espinosa et al. with foamed scaffolds, yielding as well structures with virtually inexistent interfaces.^[5a] Once we established a joining method between the solid phases of the niche-inspired scaffolds, we fabricated the scaffolds as individual compartments that were then merged together (Figure 1A–D). First, two individual 3D printed scaffolds were fabricated and shaped into a cylinder (chondrogenic phase) and a ring (osteogenic phase). The chondrogenic and osteogenic compartments were functionalized with peptide sequences derived from TGF-β1 binding epitope and BMP2 growth factors, respectively. Then, they were merged together and an ESP fibrous mesh with the shape of a cup was inserted in the bony compartment of the 3DP scaffold, and adhered as described previously. Finally, a hydrogel was formed inside the ESP cup by dropping a 1% w/v RGD-functionalized alginate solution inside and cross-linking it in a 0.1 m CaCl₂ bath. The ESP cup was fabricated longer than the cavity in the bony compartment, where it was inserted to facilitate its introduction. Regardless, the hydrogel was included solely on the bony compartment, remaining the rest of the ESP cup empty.

Figure 1. Fabrication and appearance of niche-inspired scaffolds.

A) Schematic representation of the assembly process of niche-inspired scaffolds where (1) two 3D printed structures for the chondrogenic and osteogenic phases modified with the respective peptide sequences are joined by slight dissolution of the interface followed by pressure bonding; (2) the interface of the bony cavity is dissolved by wetting and an electrospun (ESP) cup-shaped mesh is introduced and bond by pressing against the walls with the aid of a blunt needle; (3) a hydrogel solution is added and crosslinked inside the ESP cup. (4) The scaffold cross-section shows the location of the hydrogel and cup, inserted in the bony compartment of the niche-inspired scaffold. B–D) Scanning electron microscopy (SEM) micrographs of model scaffolds showing the intermediate steps of the fabrication process; B) merged osteogenic and chondrogenic 3D printed parts, with a cup-shaped ESP mesh (C), after forming a hydrogel core (D). Scale bars are 1 mm. E) Representative stress– strain traces of the niche-inspired scaffolds under compression and F) zoom-in of the elastic region of the test. G) Young’s modulus (brown), yield stress (maroon), and yield strain (green) of niche-inspired scaffolds as calculated from compression tests. Data is shown as average ± SD, n = 3.

The mechanical properties of the niche-inspired scaffolds were measured under compression displaying a response characteristic of porous scaffolds with an initial elastic region followed by a plastic deformation after approximately 10% of strain (Figure 1E–G). The plastic deformation was attributed to the collapse of the individual layers of the 3DP scaffold as indicated by the small step-like stress–strain traces at deformations above 10% approximately (Figure 1E). Notably, niche-inspired scaffolds acted as a homogeneous construct, without apparent delamination between phases. The acellular niche-inspired scaffolds presented a Young’s modulus of 55.8 ± 14.6 MPa, a yield stress of 6.7 ± 0.5 MPa and a yield strain of 9.9 ± 0.5%. The Young’s modulus of the osteochondral interface is a combination of those of cartilage, calcified cartilage, and subchondral bone, accounting for 60%, 10%, and 30% of the tissues height, respectively. The Young’s moduli of human articular cartilage is typically set between 0.3 and 0.8 MPa^[17] and it is expected to deform upon compressional loads, transferring these later to the subchondral bone with a Young’s modulus of about 20 GPa.^[18]

2.2. Niche-Inspired Scaffolds are a Depot of Nonproliferative Cells and Regulate Spatially Cell Proliferation

After fabrication and characterization of niche-inspired scaffolds, their capability to control spatially cell proliferation was assessed. Niche-inspired scaffolds were fabricated with cell-laden alginate-RGD hydrogels with or without bearing growth factor-derived peptide sequences on the 3DP compartments and compared to cell-laden alginate-RGD hydrogels alone (Figure 2). The niche-inspired scaffolds were cultured in four different media conditions: basal, chondrogenic, osteogenic, and 1:1 mixture of chondrogenic and osteogenic media, representative of their natural environment. Cell proliferation is characterized by the synthesis of new DNA when cells are in the S-phase of their cycle. After 21 days of culture, cell proliferation was evaluated by staining with 5-Ethynyl-2′-deoxyuridine (EdU), an analogue of thymidine, that readily incorporates into newly formed DNA. Cells were still present in the core of the niche-inspired scaffolds, acting as a cell depot, regardless of the culture media used. A decreased number of cells was detected in basal, osteogenic, and 1:1 media conditions as compared to chondrogenic media in niche-inspired scaffolds bearing peptide sequences (Figure 2, + peptide). A lower degree of proliferation and migration out of the hydrogel is expected during chondrogenic differentiation processes, and thus this was an expected observation. In niche-inspired scaffolds alone, without peptide sequences in the 3DP compartment, the number of cells appeared to be lower also in scaffolds cultured in chondrogenic medium, suggesting a synergetic influence of the peptides and culture media on cell migration (Figure 2, –peptide). In all niche-inspired scaffold’s core hydrogel, cells displayed a nonproliferative character as evidence by the absence of EdU⁺ cells, which suggested the induction of a quiescent-like behavior in hBMSCs as reported earlier by us.^[15] Indeed, we have previously shown that hBMSCs cultured inside these hydrogels enter the G0-phase and are able to re-enter the cell cycle when recovered from the gel and seeded in traditional plastic culture plates, enhancing their differentiation potential. Moreover, comparison to cells in alginate-RGD hydrogels alone cultured in proliferative osteogenic and mix media conditions suggest that having the hydrogel as a core of a multicompartment scaffold might reduce the availability of nutrients or decrease the oxygen concentration, thus affecting the proliferative capacity of hBMSCs (Figure 2, alginate-RGD). Control alginate hydrogels showed proliferating cells when cultured in osteogenic or 1:1 media conditions, suggesting a direct effect of these media type. In fact, strategies to induce cell quiescence have been limited to cultures in starvation media conditions (without serum), cultures with induced loss of adhesion or by contact inhibition.^[19] The alginate gel of our niche-inspired scaffolds is inherently adhesive, as designed by the introduction of RGD motives in the polymer chain. Moreover, cells in only alginate-RGD scaffolds showed a proliferative character when cultured in osteogenic and 1:1 mix media conditions. However, they remained nonproliferative in basal and chondrogenic conditions, which indicates it to be independent of the presence of FBS (with FBS in basal conditions and without in chondrogenic). Thus, our data suggest the induction of quiescence in our alginate-RGD gels to be independent of the FBS presence (starvation), adhesion, or contact inhibition processes.

Figure 2.

Niche-inspired scaffolds are a depot of nonproliferative cells. Laser scanning confocal microscopy images of host bone-marrow derived stem cells (hBMSCs) embedded in the core alginate-RGD hydrogels of niche-inspired scaffolds, with or without bearing growth factor-derived peptide sequences on the 3D printed (3DP) compartment and, control alginate-RGD scaffolds alone after 21 days of culture in basal, osteogenic, chondrogenic, and 1:1 osteogenic:chondrogenic media. Cells were stained for DNA (Hoescht, blue) and for newly synthesized DNA or proliferative activity (EdU, red). Scale bar is 100 μm. Insets in the last column are 70 × 70 μm. White arrows point at EdU⁺ cells.

When cultured for 5 weeks in basal media conditions, further investigation of cell responses via scanning electron microscopy (SEM) revealed that, indeed, cells from the hydrogel depot were able to migrate out of the hydrogel, to proliferate as indicated by the elongated shape (colored in pink) in the ESP phase, and finally to invade the 3DP compartment (Figure 3). One week after seeding, niche-inspired scaffolds (all bearing growth factor-derived peptide sequences) showed the presence of cells inside the hydrogel compartment and at the interface of the ESP mesh and the hydrogel (Figure 3, hydrocup). However, no cell migration was observed to the 3DP. Upon culture, the morphology of the cells in the hydrogel remained mostly rounded (colored blue), with few cells accounting for a spindle morphology, which could be an indication of their commitment to a nonproliferative and quiescent state. Further, observation of niche-inspired scaffolds allowed us to conclude that cells started to migrate through the ESP mesh reaching the outer layer. Here, the cells present a more elongated morphology (Figure 3, ESP), characteristic of proliferating and migrating cells. It is important to note that, even if the ESP cup has one open end where cells from the hydrogel could potentially migrate out, this was not observed during the culture time.

Figure 3.

Niche-inspired scaffolds regulate spatially cell proliferation. Scanning electron microscopy (SEM) micrographs of host bone-marrow derived stem cells (hBMSCs) in niche-inspired scaffolds cultured for 5 weeks in basal medium. Cells present in the alginate-RGD hydrogel compartment remained mostly rounded (colored in blue), with few elongated cells (colored in pink), and were able to migrate to the hydrogel-electrospun (ESP) cup interface (hydrocup), acquiring then an elongated morphology in the ESP mesh. Finally, the cells proliferated and invaded the 3D printed (3DP) compartment bearing growth factor-derived peptide sequences after 5 weeks of culture. Scale bars are 1 mm for niche-inspired scaffold micrographs, 200 μm for hydrocup micrographs, 20 μm for hydrogel and ESP micrographs, and 50 μm for the 3DP micrographs. Original noncolored images can be found in Figure S3 (Supporting Information).

Finally, observation of the pores of the 3DP compartment proved that the entire scaffolds was populated by cells. After only 2 weeks of culture, cells were observed bridging the perimeter of the pore walls. Upon culture time, the cells invaded the pores, filling them up after 5 weeks (Figure 3, 3DP).

ESP scaffolds have been intensively explored in the literature to manipulate cell migration and differentiation.^[20] Due to their fibrous ECM-like nature, ESP scaffolds proved to induce a higher proliferation rate and faster migration of hBMSCs than cell culture plates.^[21] Comparison between meshes with different fiber diameters (namely, 400, 800, and 1200 nm) has shown that thinner diameters promote a higher proliferation rate and faster migration across the surface of the mesh. Although our ESP accounts for a fiber diameter of 2.9 ± 0.2 μm that could result in a slower migration speed across the surface, higher fiber diameter forms a mesh with bigger pores, facilitating cell migration through the cross-section of the mesh or cell infiltration.^[22]

2.3. Niche-Inspired Scaffolds Drive the Deposition of a Zone-Specific Matrix

An important feature of scaffolds aimed at the regeneration of the osteochondral interface is the capability to support zone-specific—cartilage and subchondral bone—tissue formation. To evaluate the capability of our niche-inspired scaffolds, and particularly of our peptide functionalized 3DP structure to selectively drive cell differentiation and tissue formation, we cultured hBM-SCs embedded within the hydrogel compartment of the niche-inspired scaffolds in the different types of media (namely, basal, chondrogenic, osteogenic, and 1:1 compositions) (Figure 4). After 3 weeks of culture in the designated media, we further investigated the formation of calcium deposits and the deposition of glycosaminoglycans (GAGs) via histological staining with Alizarin Red and Safranin-O, respectively (Figure 4A). After 3 weeks of culture, +peptide scaffolds showed a higher amount of calcium deposition compared to −peptide scaffolds, independently of the media used for cell culture. Moreover, the staining appeared to be more intense on the bone side (bottom phase of the scaffold, labeled B) in all media conditions, suggesting a direct effect of the grafted BMP-2 derived peptide. GAG deposition revealed a weak pink stain in functionalized scaffolds. This could be explained as the result of a higher cell invasion in the 3DP compartment, also observed by SEM (Figure S4, Supporting Information), where the cells stained in dark brown (iron hematoxylin) covered the GAG staining. Nevertheless, the localization of GAGs appeared to be more intense in the upper half of the scaffold (labeled as C) corresponding to the cartilage region particularly for the scaffolds cultured in basal and chondrogenic media conditions. This could be an indication of the effect of the grafted chondrogenic peptide in niche-inspired scaffolds.

Figure 4. Niche-inspired scaffolds promote osteochondral-like tissue formation.

A) Histological evaluation of calcium deposits (Alizarin Red, red) and deposition of glycosaminoglycans (GAGs) (Safranin-O, pink) in the niche-inspired scaffolds after 3 weeks of culture in basal, osteogenic, chondrogenic, or mix media conditions in functionalized (+peptide) or bare (–peptide) scaffolds. Scale bar is 500 μm. Cells in Safranin-O staining were counterstained with iron hematoxylin (dark brown). B) Cell number and DNA normalized C) alkaline phosphatase (ALP) activity and D) GAG and E) osteocalcin deposition after 3 weeks of culture on niche-inspired (+ and – peptide, blue and red bars, respectively) scaffolds cultured in basic, osteogenic, chondrogenic or mix media. H) Young’s modulus of niche-inspired scaffolds after 3 weeks of culture on the different scaffolds and media conditions. n = 3 for all experiments. Data is shown as average ± standard deviation. Significance in B–F was calculated with a two-way ANOVA with Sidak’s multiple comparison test; ****p < 0.0001, ***p < 0.0002, **p < 0.0021, and *p < 0.0332. For DNA: interaction F (5, 24) = 8.572, p < 0.0001; media type F (5, 24) = 11.07, p < 0.0001; peptide F (1, 24) = 35.84, p < 0.0001. For ALP: interaction F (4, 20) = 0.1139, p = 0.9761; media type F (4, 20) = 43.63, p < 0.0001; peptide F (1, 20) = 0.5336, p = 0.4735. For GAGs: interaction F (4, 20) = 36.80, p < 0.0001; media type F (4, 20) = 52.25, p < 0.0001; peptide F (1, 20) = 90.94, p < 0.0001. For osteocalcin: interaction F (3, 16) = 18.63, p < 0.0001; media type F (3, 16) = 30.93, p < 0.0001; peptide F (1, 16) = 29.10, p < 0.0001. For Young’s modulus: interaction F (3, 16) = 7.299, p = 0.0027; media type F (3, 16) = 9.239, p = 0.0009; peptide F (1, 16) = 10.18, p = 0.0057.

The cell number was quantified from the different scaffolds and media conditions (Figure 4B). A decrease of the cell number was observed for all the media conditions when the 3DP scaffold was bearing peptide sequences (blue bars, +peptide). Conversely, cells cultured in scaffolds with no peptide (red bars, −peptide) in osteogenic and mix media conditions were able to proliferate. This result could be interpreted as an indication of the phenotypic state of the cells. While cells on peptide bearing scaffolds differentiated and dedicated to the synthesis of new extracellular matrix (ECM), cells on the −peptide scaffolds divided, and therefore did not deposit ECM. Samples cultured in chondrogenic medium showed a decrease on the cell number as compared to the initially seeded number of cells. It is, however, known that cells in chondrogenic conditions or within cartilage are resting, nonproliferative cells. It is important to note that there was no significant difference between the amounts of seeded cells (day 0) and the cell number after 24 h of culture, indicating that the alginate cross-linking step did not affect cell viability.

The formation of an ECM characteristic of chondrogenic and osteogenic environments was evaluated by measuring the alkaline phosphatase (ALP) activity (bone) and deposition of GAGs (cartilage and bone) and osteocalcin (bone) (Figure 4C–E). For all media compositions, the deposition of GAGs per DNA after 3 weeks of culture was higher for hBMSCs cultured in peptide bearing scaffolds as compared to the nonfunctionalized counterparts (Figure 4D). This effect was particularly noticeable for basic and osteogenic media conditions. This result aligns with our previous hypothesis where we expected the cells in nonfunctionalized scaffolds to deposit less ECM as result of being in a more proliferative state. hBMSCs cultured on functionalized scaffolds in osteogenic medium presented a GAG deposition per DNA that was similar (not significantly different) to that one deposited by hBMSCs in both functionalized or nonfunctionalized scaffolds in chondrogenic conditions. This could be an indication of media driven GAG deposition when cells are cultured on chondrogenic medium conditions. Nevertheless, the total GAG deposition was not significantly different across media or peptide conditions when the DNA (or total cell number) content was not taken into consideration (Figure S5, Supporting Information).

The expression of ALP was measured on cells cultured in all media conditions after 3 weeks of culture. A clear increase in ALP activity (Figure S5, Supporting Information) and DNA normalized ALP activity (Figure 4C) of two-orders of magnitude was measured for samples cultured in osteogenic medium. However, no significant difference was detected between +peptide and −peptide scaffolds, indicating a media driven deposition of ALP or differentiation of hBMSCs. No ALP expression increase was observed for the basic, chondrogenic, or mix media conditions.

The release of osteocalcin to the media was also measured (Figure 4E), showing no significant difference between osteogenic, chondrogenic, and mixed media conditions on scaffolds with or without peptides after 3 weeks of culture. Osteocalcin is a late marker of differentiation, expressed by mature osteoblasts, and thus it would be expressed at later time-points.

Niche-inspired scaffolds proved the capability to induce a nonproliferative character in cultured hBMSCs and serve as cell-depot (Figure 2), to induce migration and in vasion of the 3DP compartment when cultured in basal medium (Figure 3) and to induce the deposition of location-specific proteins (Figure 4). Next, we evaluated their capability to induce hBMSC migration and deposition of ECM when cultured in the various tissue-specific media conditions (Figure S4, Supporting Information). After 3 weeks of culture, all the niche-inspired scaffolds retained their shape with characteristic regions for the 3DP, ESP, and hydrogel. Within the ESP region hBMSCs appeared to be spread in osteogenic media (+ and –peptide) and in the chondrogenic media when the scaffold is not functionalized (−peptide). In the mix condition, a mixed population of hBMSCs is visible with some spread cells and some others accounting for a rather rounded morphology. hBMSCs cultured in basal conditions (+ and –peptide) appeared rounded, as well as in chondrogenic and mix media where the scaffolds are functionalized with peptides (+ peptide). Despite this morphometric observation, hBM-SCs in basal and mix media conditions (+ and –peptide) and in osteogenic media where the scaffolds were functionalized (+ peptide), were able to migrate out of the hydrocup, and populate the 3DP compartment of the scaffold. hBMSCs cultured on chondrogenic medium were not able to populate the 3DP compartment. This is expected as chondrogenic differentiation reduces cell proliferation. Taking into account the measured number of cells (Figure 4A), we can conclude that proliferation and migration occur as separate events within the niche-inspired scaffolds. Thus, in basal and osteogenic conditions, despite accounting for a higher cell number when the scaffolds are nonfunctionalized, these cells are mainly located within the hydrocup, whereas on the functionalized counterparts cells migrate and populate the 3DP compartment, which we ascribed to haptokinetic effects, as previously shown by us with other scaffolds-bound peptide sequences.^[23]

Together with cell invasion and matrix deposition, increased mechanical properties are generally expected if material degradation does not occur first. During material degradation, however, degradation should come together with tissue formation, enabling load bearing in vivo.^[24] The mechanical properties of the niche-inspired scaffolds were measured under compression in liquid after 3 weeks of culture in all media conditions (Figure 4F and Figure S4, Supporting Information). The stress– strain curves showed an initial linear regime corresponding to the elastic region, followed by an exponential increase due to the densification of the porous material and a final plateau corresponding to a stepwise failure of the structure. Indeed, a step was observed for every layer of the scaffold breaking as in the mechanical tests of scaffolds prior to cell culture (Figure 1E–G). The Young’s modulus was measured from the elastic region of the stress–strain curves (Figure 4F). Overall, the scaffolds showed a Young’s modulus that oscillates between 40 and 70 MPa. The lowest Young’s moduli were recorded for −peptide scaffolds and scaffolds cultured in mix media conditions with values of 40.4 ± 3.9; 40.6 ± 6.9; 37.9 ± 8.7; and 51.0 ± 4.5 MPa for osteo-peptide, chondro-peptide, mix +peptide, and mix-peptide conditions, respectively. This suggests a lower amount of ECM being deposited in these scaffolds as previously observed by SEM (Figure S3, Supporting Information). Contrary, the mechanical properties of the scaffolds +peptide and scaffolds cultured in basic media were highest with values of 72.8 ± 11.5; 65.7 ± 12.4; 71.6 ± 9.3; and 59.8 ± 6.9 MPa for basic+peptide, basic-peptide, osteo+peptide, and chondro+peptide, respectively. Interestingly, the Young’s modulus of niche-inspired scaffolds seem to be reduced during the culture period as compared to initial scaffolds (E’ = 55.8 ± 14.6 MPa, Figure 1), as evidenced in −peptide and mix media culture conditions. However, in +peptide and basal conditions the Young’s moduli increased, suggesting a compensation of the softening of the scaffolds with the new matrix being deposited in niche-inspired scaffolds bearing peptide sequences.

2.4. Niche-Inspired Scaffolds Drive hBMSCs Differentiation

The phenotypic state of the cells was studied after 3 weeks of culture to further demonstrate the differentiation induction potential of the peptide-functionalized 3DP scaffolds on infiltrated cells. As control, niche-inspired scaffolds without peptide functionalization were used (Figure 5). Gene expression was studied on the entire population of cells within the scaffolds as delamination of the niche-inspired scaffolds was not possible. We studied the gene expression of the Sox trio (5, 6, and 9), RunX2 and SP7 (Osterix) transcription factors as well as BSP (bone sialoprotein) and collagens I, II, and X as characteristic markers for chondrogenic and osteogenic differentiation. The transcription factor Sox9 works together with Sox5 and Sox6 to regulate the expression of col2a1, an important marker for chondrogenesis. When only Sox9 is expressed within the trio, and appears associated to gli2/3, it can suppress the transcription of col10a1, but can also lead to collagen I and osteopontin production, being the latter both osteogenic markers.^[25] Gene expression analysis showed an upregulation of Sox5 and Sox6 for all scaffolds and media conditions. Sox9 was downregulated for osteogenic and mix conditions and upregulated in chondrogenic media. However, in our experience, Sox9 is highly expressed on in vitro expanded hBMSCs. Although being downregulated as compared to cells before the experiment, the relative expression of this marker was still high.

Figure 5.

Niche-inspired scaffolds support host bone-marrow derived stem cells (hBMSC) differentiation. Gene fold increase expression of Sox5, Sox6, Sox9, RunX2, collagen I, collagen II, collagen X, and bone sialoprotein (BSP) in hBMSCs cultured and in +/− peptide scaffolds in all different media conditions for 3 weeks. Data is presented as fold change relative to hBMSCs before the experiment. Relative gene expression of SP7 in all scaffold and media conditions after 3 weeks of culture. Significance was calculated with a two-way ANOVA with Sidak’s multiple comparison test; ****p < 0.0001, ***p < 0.0002, **p < 0.0021, and *p < 0.0332. F) For Sox5: interaction F (3, 16) = 0.3275, p = 0.800534; media type F (3, 16) = 4.876, p = 0.013533; peptide F (1, 16) = 0.1263, p = 0.726965. For Sox6: interaction F (3, 16) = 12.76, p = 0.000164; media type F (3, 16) = 27.96, p = 0.000001; peptide F (1, 16) = 17.30, p = 0.000739. For Sox 9: interaction F (3, 16) = 0.6600, p = 0.588525; media type F (3, 16) = 19.08, p = 0.000016; peptide F (1, 16) = 0.3141, p = 0.582943; For RunX2: interaction F (3, 16) = 0.4956, p = 0.690461; media type F (3, 16) = 10.43, p = 0.000479; peptide F (1, 16) = 3.164, p = 0.094280. For collagen I: interaction F (3, 16) = 6.444, p = 0.004561; media type F (3, 16) = 28.51, p = 0.000001; peptide F (1, 16) = 6.306, p = 0.023147. For collagen II: interaction F (3, 14) = 32.15, p = 0.000002, media type F (3, 14) = 354.0, p<0.000001; peptide F (1, 14) = 2.933, p = 0.108829. For collagen X: interaction F (3, 16) = 7.110, p = 0.002990; media type F (3, 16) = 37.03, p<0.000001; peptide F (1, 16) = 7.191, p = 0.016379. For BSP: interaction F (3, 11) = 1.599, p = 0.245677; media type F (3, 11) = 15.67, p = 0.000276; peptide F (1, 11) = 1.836, p = 0.202637. For SP7: interaction F (3, 14) = 1.306, p = 0.311428; media type F (3, 14) = 27.35, p = 0.000004; F (1, 14) = 0.3055, p = 0.589163.

These data correlated to the measured gene expression of collagen I that was upregulated 4–7 fold in osteogenic, chondrogenic and mix conditions. Moreover, cells cultured on peptide bearing scaffolds presented a higher expression of collagen I than their nonfunctionalized counterparts (−peptide), suggesting the functionality of the grafted peptides. Collagen II expression was not detected on basal or mix conditions, but it was 10-fold upregulated in hBMSCs cultured in peptide bearing scaffolds cultured in chondrogenic media. Moreover, this upregulation was three-fold higher than for cells in nonfunctionalized scaffolds also in chondrogenic media. We interpreted this as an indication of the effects of peptide functionalization in the scaffolds. Collagen X, a protein expressed by osteoblast or hypertrophic chondrocytes was also upregulated in all media conditions, accounting for a significantly higher expression in nonfunctionalized scaffolds cultured in chondrogenic and mix conditions. A general overexpression of collagens is generally expected during hBMSC differentiation as traditional media contains ascorbate 2-phosphate, which is known to promote protein synthesis.

We also analyzed the expression of the transcription factor RunX2, generally associated to osteoblast differentiation. A 2– 4 fold upregulation was measured for hBMSCs cultured in all media conditions, independently of the type of scaffold used (+peptide and −peptide). RunX2 is a transcription marker expressed in cells that are already committed toward osteogenic phenotypes. However, the transcription factor has been proposed to be a regulator of the cell cycle. In this case, RunX2 interacts with p53 and cyclins D2 and D3 regulating the cell cycle progression with maximum levels of RunX2 being expressed at G0/G1 phase transition of the cell cycle.^[26] Thus, perhaps indicating a re-entry into the cell cycle, progression, and proliferation.

Together with RunX2, another major regulator of the mesenchymal to osteoblastic regulator is the transcription factor SP7 or osterix. Indeed, SP7 expression was not detected in hBMSCs prior encapsulation into niche-inspired scaffolds. However, relative expression analysis of the gene in hBMSCs cultured in the different media conditions showed a higher expression in cells cultured in osteogenic media, independently of the surface functionalization of the scaffold, followed by basic media conditions. Relative expression in mix media (+peptide and −peptide) and +peptide in chondrogenic media were negligible, while a low expression was detected on cells cultured in chondrogenic media in nonfunctionalized scaffolds (−peptide). SP7 is expressed in mature osteoblasts and acts as regulator of chondrogenesis, inhibiting chondrocyte maturation and promoting osteoblast maturation during osteochondral ossification. Thus, it appears that, when cultured in osteogenic media, an osteoblastic differentiation was promoted, and chondrocyte maturation was most likely inhibited in chondrogenic media conditions of +peptide scaffolds. On the other hand, BSP was upregulated in all media conditions, being more pronounced in chondrogenic medium, particularly in nonfunctionalized, −peptide, scaffolds. It is well-known that chondrogenesis in vitro results in the progression of hBMSCs toward hypertrophic chondrocyte lineages and this result might therefore suggest that the inclusion of a TGF-β derived peptide sequence might be inhibiting—at least partially—this effect.

The peptide sequence derived from the binding epitope of TGF-β1 used here has been shown to increase in vitro the expression of aggrecan and Sox-9 on hBMSCs cultured in supramolecular hydrogels bearing this sequence, resulting also on a higher deposition of GAGs.^[27] Previous studies from our group using this TGF-β1 derived sequences in 3DP scaffolds showed also an upregulation of Sox9, collagen II and collagen X, validating its functionality when “clicked” in this type of stiff materials.^[6a]

Here, we observed a higher upregulation of collagen II when cultured in chondrogenic medium as compared to our previous studies, although we have a combination of an osteogenic and a chondrogenic peptide sequence. Similarly, the peptide sequence derived from BMP-2 binding domain used here was previously shown to enhance the expression of Smad in hBMSCs, an osteoblastic differentiation marker.^[28] However, in our previous studies, analysis of the gene expression of hBMSCs cultured in 3DP scaffolds bearing this peptide sequence failed to show a significant upregulation of osteogenic markers as compared to nonfunctionalized scaffolds. Here, hBMSCs cultured in osteogenic medium and scaffolds bearing the BMP-2 and TGF-β1derived peptide sequences expressed collagen I to a higher degree than nonfunctionalized counterparts, suggesting an increased differentiation potential of hBMSCs in niche-inspired scaffolds. We hypothesized that this enhanced differentiation effect is a result of the cell-depot of niche-inspired scaffolds that present a nonproliferative, “quiescence-like” character. Thus, hBMSCs in niche-inspired scaffolds would be all synchronized in the cell cycle and present a greater stemness that enhanced their chondrogenic potential.

Overall gene expression analysis showed a higher dependence on the media type used (significant for all gene expression analysis). Interestingly, the influence of the inclusion of the peptides was significantly different for the expression of collagen I when cultured in osteogenic medium, being higher for +peptide conditions. The presence of peptides was also significant on the expression of collagen II, which was higher in hBMSCs cultured in chondrogenic media in +peptide scaffolds and osteogenic media in −peptide scaffolds. Furthermore, the inclusion of the peptides also influenced the expression of collagen X, showing a higher expression on −peptide scaffolds in chondrogenic medium. This overall indicates a direct effect of the peptide on matrix deposition proteins characteristic of chondrogenic and osteogenic environments.

2.5. Biocompatibility and Tissue Formation in Rat Subcutaneous Models

After investigating the potential of niche-inspired scaffolds to serve as cell depot and to steer the selective differentiation of hBMSCs, in vivo biocompatibility was evaluated in a preliminary rat subcutaneous model. Full niche-inspired scaffolds and the individual components were separately implanted (Figures S7–S9, Supporting Information). After 4 weeks of implantation, the samples were harvested and analyzed via histological staining. 3DP scaffolds were tested on their peptide functionalized (+peptide) and nonfunctionalized (−peptide) versions. 3DP scaffolds appeared to be well integrated on the skin and no encapsulation or acute inflammatory response was visible (Figure S7H&E, Supporting Information). Detailed observation revealed a good integration of not only the scaffolds as a whole but also the individual fibers of these ones. Cells infiltrated the functionalized as well as the nonfunctionalized scaffolds. Masson’s trichrome staining revealed the deposition of a collagenous matrix in both functionalized and nonfunctionalized samples, as evidenced by a blue staining, with an apparently higher collagen deposition in +peptide scaffolds. Similarly, H&E showed a higher amount of matrix and cells in +peptide scaffolds, although quantitative analysis was not performed. Safranin-O staining revealed a higher deposition of GAGs in functionalized samples (+peptide), as observed by the more purple colour of the deposited matrix.

To study the biocompatibility of the ESP component and its capability of allowing cell migration and infiltration through the scaffold, cell free samples were prepared in two formats, thinner (300 × 8 mm) and thicker ESP discs (600 × 8 mm) (Figure S8, Supporting Information). All scaffolds significantly increased their cross-sectional dimensions to 1046 ± 119 μm for the thin format and 1576 ± 285 μm for the thick version. ESP scaffolds integrated well with the surrounding tissue and no inflammatory response or encapsulation was observed (H&E). A detailed observation with Masson’s trichrome revealed a higher cell infiltration in thinner scaffolds compared to thicker ones, with 8.5 ± 2.2% of the section being infiltrated as compared to 5.3 ± 0.3%, respectively. However, no matrix deposition was observed within the bulk of the ESP. We designed the niche-inspired scaffolds with an ESP component of 75 μm wall thickness as a migratory and proliferative phase, and thus this observation was considered positively for the functionality of the full scaffolds. Safranin-O staining presented an intense pink staining that we ascribed to the polymer itself and not to the deposition of GAGs.

Finally, we investigated the biocompatibility of the hydrogel and its integrity after 4 weeks of implantation. Alginate gelation occurs via ionic cross-linking and the encapsulation of cells results in a decreased number of cross-linking points, and therefore integrity over time. Thus, we investigated the alginate-RGD hydrogel when loaded with or without cells (Figure S9, Supporting Information). After 4 weeks of implantation, the hydrogels were well-integrated and no inflammatory response was observed. Hydrogels alone appeared more cohesive, with nondisrupted sections and a slight tissue accumulation on the perimeter of the hydrogel. It is noteworthy that the hydrogel sections were positively stained, showing the background polymer. hBMSC containing hydrogels, on the other hand, appeared more disrupted, but also more integrated within the tissue and cells were still visible within. Masson’s trichrome staining made evident the presence of a thin collagenous layer surrounding the hydrogels (w/o cells) and showed the presence of cells within the cell-seeded hydrogels. No deposition of GAGs was observed within the hydrogels other than the background stain. This observation appears to support our previous data where the cell depot inside hydrogels remained nonproliferative and formation of matrix was not expected.^[15]

Study of the biocompatibility on the full niche-inspired scaffolds was performed on peptide-functionalized scaffolds with and without cells encapsulated within the hydrogel (Figure S10, Supporting Information). No scaffold encapsulation or inflammatory response was detected from Masson’s trichrome staining. Moreover, the scaffolds were well integrated and showed clear cell infiltration filling up the 3DP compartment. Detailed images of the ESP/3DP interface (Figure S10, Supporting Information, bottom row) revealed the presence of cells also within the most outer layer of the ESP, indicating cell migration from the hydrogel and throughout the ESP phase. Masson’s trichrome confirmed the deposition of a collagenous matrix in both sample types (with or without cells).

In a second in vivo study, the capability of the niche-inspired scaffolds to drive the formation of a neo-tissue on a subcutaneous implantation model in rats was conducted. Samples were harvested after 3 (Figure S11, Supporting Information) and 6 weeks (Figure 6) of implantation and tissue formation was evaluated by means of histological staining with H&E and Masson’s trichrome. Samples were prepared with or without functionalization (+ or –peptide) and also with and without cells to be able to evaluate the effects of these parameters on the tissue formation. For niche-inspired scaffolds with cells, these were only located inside the hydrogel and not in the 3DP structure.

Figure 6.

In vivo tissue formation in rat subcutaneous models. Optimal microscopy images of tissue sections after 6 weeks of implantation of the hydrocup, hydrogel, 3D printed (3DP) and the full niche-inspired scaffolds in their peptide functionalized (+peptide) and nonfunctionalized (−peptide) versions with or without host bone-marrow derived stem cells (hBMSCs) seeded in the scaffolds 24 h prior implantation and stained with Masson’s trichrome or hematoxylin & eosin (H&E). Arrows point at the electospun/3DP interface where cell migration occurs. Scale bar in the individual compartments (hydrocup, hydrogel, 3DP) is 1 mm. Scale bars in all images of niche-inspired scaffolds is 500 μm.

After 6 weeks of implantation, H&E staining revealed a good integration of the scaffolds with no encapsulation or apparent chronic inflammatory response (Figure 6). The individual hydrocup scaffolds appeared to have a greater cell and tissue infiltration as compared to 4-week implantations, independently of the chemistry of the hydrogel within and presence or absence of cells. Detailed observation revealed the presence of the implanted cells still residing in the hydrogel phase. The hydrogels implanted without cells and without peptides appeared to retain integrity only when no cells were embedded within, probably due to the lower amount of cross-linking points formed when cells or peptides were included within. Cells were not detected inside the hydrogels, probably due to the faster disintegration of the structure that would allow cells to migrate out. 3DP scaffolds appeared to be fully integrated with a great amount of collagen deposition, as observed by Masson’s trichrome staining, when they were preseeded with cells and independently of the chemistry. 3DP scaffolds without cells were also infiltrated and the deposition of a collagenous matrix was also present, but not yet filling the entire scaffolds. Similarly, niche-inspired scaffolds started to be infiltrated, independently of the chemistry of the scaffold and the presence of seeded cells. Detailed observation of the scaffolds on high magnification images showed a good interface between the ESP and the 3DP compartments, with cells in close contact with the hydrocup. This was visible in all scaffolds and cell conditions, suggesting that even when cells were not seeded in the hydrogel, host cells were able to populate the 3DP scaffolds from outside in. It is also important to note that cell migration occurred in both directions from the hydrogel outside through the ESP and from the host into the ESP. This effect can be visualized on high magnification images on samples that were not preseeded, as a darker staining on the ESP most outer layers (Figure 6, black arrows).

2.6. Niche-Inspired Scaffolds Regenerate the Osteochondral Interface

Finally, niche-inspired scaffolds were evaluated for their potential to regenerate the osteochondral interface in vivo in a rabbit osteochondral critical size defect model. Niche-inspired scaffolds were prepared with cell-laden RGD-functionalized hydrogels (Figure 7, scaffold w/cells) and with RGD-functionalized hydrogels without cells (Figure 7, scaffold) and compared to control groups of nontreated defects (Figure 7). Here, 3DP compartments were fabricated continuously, without dissolution blending, using a homemade hybrid printer presented elsewhere.^[6a,29] Control groups showed the regeneration of cartilage with columnar arrangement of chondrocytes that appeared to be grouped forming lacunae. In addition, the formation of a clear tidemark was observed. The integration with the existing healthy cartilage was, however, poor as can be observed by Masson’s trichrome and H&E staining (Figure 7, control, interface). The deposition of collagens and GAGs appeared to be lower (faint stain) on the regenerated zone as compared to the surrounding tissue. However, it is worth to note that critical-size defects in control groups regenerated surprisingly good, with an overall O’Driscoll score of 20.7 ± 1.6. Evaluation of the regenerated cartilage after the implantation of niche-inspired scaffolds and cell-laden niche-inspired scaffolds showed a clear deposition of connective tissue that was higher (more intense reddish stain, Figure 7A, Masson’s trichrome, cartilage) for cell-laden scaffolds. Contrary, the deposition of GAGs observed by Safranin-O staining appeared to be more intense in scaffolds without cells. Yet, cellladen niche-inspired scaffolds had a similar appearance as control regenerated cartilage. The tidemark formation was not very clear in both scaffolds types, yet improved in cell-laden scaffolds (Figure 7A, cartilage/bone). This is probably partially hindered by the scaffold itself; further degradation of the scaffolds is expected to lead to further tissue remodeling and better tidemark area formation. Bone formation was more prevalent in cell-laden scaffolds, suggesting a direct effect of the cell depot present in niche-inspired scaffolds, as observed by collagen deposition (stained in blue, Masson’s trichrome). Basal integration of the scaffolds was not affected by the type of implanted scaffolds.

Figure 7. Osteochondral regeneration in rabbit defect models.

A) Optimal microscopy images of tissue sections of the osteochondral interface (overview) of untreated rabbit knees after defect formation (control), niche-inspired scaffolds without cells (scaffold) and niche-inspired scaffolds with cell-laden hydrogels (scaffold w/cells) after 12 weeks of implantation and stained with Masson’s trichrome (connective tissue in red, collagen in blue and nuclei in black) and Safranin-O (cartilage in orange to red, nuclei black, cytoplasm bluish to green). Detailed magnified images show the cartilage area, cartilage to bone interface, subchondral bone (bone), and the interface between the scaffold and the host tissue (interface). Scale bars are 100 μm. B) Evaluation of histological results through O’Driscoll and International Cartilage Repair Society (ICRS) macroscopic and ICRS II microscopic scores, including tissue morphology, basal integration, chondrocyte clustering and tidemark formation scores for cartilage repair. Statistical significance was calculated with one-way ANOVA with a Tukey’s post hoc test. ****p < 0.0001, ***p < 0.001, **p < 0.01, and *p < 0.1. For O’Drscoll score: interaction F = 9.564, p = 0.000770; defect treatment type: F (2, 26) = 9.564, p = 0.000770. For ICRS macroscopic score: interaction F = 34.98, p < 0.000001; defect treatment type: F (2, 29) = 34.98, p < 0.000001. For tissue morphology: interaction F = 2.729, p = 0.084006; defect treatment type F (2, 26) = 2.729, p = 0.084006. For basal integration: interaction F = 1.384, p = 0.268451; defect treatment type F (2, 26) = 1.384, p = 0.268451. For chondrocyte clustering: interaction F = 5.058, p = 0.013950; defect treatment type: F (2, 26) = 5.058, p = 0.013950. For tidemark formation: interaction F = 4.675, p = 0.018434; defect treatment type F (2, 26) = 4.675, p = 0.018434.

The regenerated cartilage was also evaluated by O’Driscoll (Table S2, Supporting Information), International Cartilage Repair Society macroscopic (ICRS) and ICRS II microscopic (Tables S3 and S4, Supporting Information) scoring systems modified from the original by Mainil-Varlet et al.^[30] Evaluation with the O’Driscoll system resulted on a score of 20.7 ± 1.6 for the control and 14.1 ± 3.5 and 13.5 ± 4.1 for the cell-free and cell-laden niche-inspired scaffolds, respectively, and being 24 the maximum grade assigned to healthy cartilage. While the O’Driscoll system reports one global score, the ICRS system evaluates the different subcategories (corresponding to the tissue microstructure) independently, assigning a score to each of them (Figure 6B and Figure S12, Supporting Information). The ICRS macroscopic score resulted on a grade IV (severely abnormal, score of 1–3) for control defects and an average of 3.4 ± 0.7 and 3.5 ± 0.7 for the cell-free and cell-laden niche-inspired scaffolds where most of the samples fall on the grade III (abnormal, score of 4–7) (Figure 7B). The ICRS II microscopic score showed a good overall tissue morphology and basal integration with no significant differences between groups. Chondrocyte clustering was significantly higher in the control group than in cell-free scaffolds but not significantly different to cell-laden niche-inspired scaffolds.

Different studies aimed at osteochondral regeneration in vivo using hydrogel-based scaffolds have shown an excellent regenerative potential,^{[3b,c,4a,b,6b]} including those bearing the TGF-β1 peptide sequence used here with a reported O’Driscoll score of 21.85 ± 1.19.^[31] Hydrogel-based scaffolds on 3D printed or injected formats hold great promise as their decomposition and clearance by the body or degradation in vivo is relatively fast, quickly leaving space for new tissue to be formed. It is noteworthy, however, that hydrogel-based scaffolds tend to have very low mechanical properties that might result in failure of the newly formed tissue in the long term. However, most in vivo studies are conducted only up to 12–16 weeks implantation where these challenges might not be evident.

Using classically stiffer thermoplastic materials such as poly(L-lactic acid), Lee et al. developed biphasic 3D printed microchambers where the fibers of the scaffold were coated with either TGF-β3 or BMP-2 growth factors to drive the differentiation of seeded adipose-derived stem cell spheroids toward chondrogenic or osteogenic phenotypes, respectively.^[3a] Implantation of spheroid-seeded chambers on rabbit osteochondral defect models showed a significant increase on the outcomes of surgery, with a score of 25 ± 3.7 over 35, with overall defect, subchondral bone, and cartilage formation being evaluated. It is important to note that, despite the increased knowledge and very interesting outcomes being reported in the field with the use of in-lab manipulated cells, these operations still hold some regulatory concerns and approval by the ethical committees for their extrapolation in clinical settings is still a persistent burden. Recently, Gong at el. reported on the fabrication of 3D printed biphasic scaffolds based on methacrylated gelatin loaded with IL-4 (interleukin-4) for the cartilage compartment and hydroxyapatite-PCL scaffold for the subchondral bone compartment).^[32] In an osteochondral rabbit defect model, after 16 weeks of implantation, they reported a modified O’Driscoll score of 24 ± 2 (over 27 points). In our study, we reported on the use of stiff PCL with a relatively long on-set of degradation (longer than the 12 weeks of implantation). Thus, despite having a good tissue integration and good tissue morphology, the O’Driscoll score was lower than those reported using hydrogels or embedded spheroids, resulting in a delayed presence of a well-defined tide-mark formation. However, an improvement of these results is expected overtime and as scaffold degradation occurs. In addition, future studies should aim at further improving the bioactivity of these Matryoshka-like niche-inspired scaffolds, for example, by further tailoring the peptide sequences attached to the 3D printed outer scaffolds compartment. Alternatively to peptides, growth factors could be considered or even more elegantly nanobodies able to sequester endogenously produced growth factors.

Clinical application of the developed scaffolds would require upscaling of the manufacturing process. Although these scaffold are complex in architecture, once the individual compartments are industrially manufactured, the assembly process would be quick and feasible at the surgical table. In fact, this process appears to be simpler than current prosthetic implants where sawing and hammering of bone is often times required. Moreover, stem cells could be directly harvested from the bone marrow or adipose tissue of the patient without further in vitro expansion, facilitating the entire process.

3. Conclusion

In summary, we designed niche-inspired scaffolds to regenerate the osteochondral interface following a nature-inspired approach with a hydrogel serving as a depot of nonproliferative cells mimicking the bone marrow, an ESP cup containing the hydrogel and serving as a proliferative phase, and a 3D printed construct with a zonal peptide-functionalizatoin to drive site-specific cell differentiation and matrix deposition. Our in vitro data showed the capability of niche-inspired scaffolds to retain the nonproliferative state of hMSCs encapsulated on the hydrogel compartment that serves as a “pool of cells” to heal the damaged area. These cells were able to migrate and populate the entire scaffolds when the 3DP scaffold was functionalized with TGF-β and BMP-2 derived peptide sequences as shown by SEM. hBMSCs cultured in these scaffolds formed calcium deposits in all conditions in the functionalized scaffolds and expressed ALP when cultured in osteogenic media. Moreover, the calcium deposition was zone dependent, showing a higher deposition on the section of the scaffold functionalized with osteogenic peptides, as shown by Alizarin Red staining. GAG deposition was also visualized histologically, showing again a zone dependent effect. A higher expression of collagens was also detected in functionalized scaffolds as compared to nonfunctionalized ones, which we ascribed to a peptide-promoted cell differentiation. In rat subcutaneous implantation models in vivo, niche-inspired scaffolds and the compartments thereof proved biocompatible and showed a substantial tissue formation with seamless interfaces. Analysis of the osteochondral regenerative potential in rabbit osteochondral defect models showed after 12 weeks of implantation a good tissue morphology, basal integration, and chondrocyte clustering. Thus, the Matryoshka-like biological construct design here is a promising new strategy for osteochondral regeneration as it combines the benefits of cell-laden hydrogels acting as a cell depot with the versatility of ESP meshes and AM scaffolds capable of providing enhanced physicochemical and mechanical properties, and tailorable bioactive domains.

4. Experimental Section

All experimental details are described in the associated Supporting Information.

Supplementary Material

Supporting Information is available from the Wiley Online Library or from the author.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.