Auricular cartilage tissue was obtained from freshly collected Capra ears by eliminating the skin with hypertonic solution, and the tissue was physically pulverized, decellularized with NaOH, and lyophilized as described in our previous works (Maity et al., 2019; Maity et al., 2022). For CDs synthesis, different concentrations (25, 50, 100, 150 and 200 µL per 20 mL of water) of HNO₃ were added to 300 mg of the lyophilized mass, and sonicated in probe sonicator (Oscar Ultrasonics, India) for 1 h followed by microwave irradiation (750 W) for 6 min. Next, the partially carbonized fractions were ground, mixed with 20 ml deionized water (diH₂O), sonicated for 1 h and pH was adjusted to 7.4. Finally, obtained CDs was dialyzed using dialysis membrane of 3.5 kDa molecular cut-off against diH₂O followed by lyophilization.

Capra auricular cartilage-derived Collagen II (COLII) was used to synthesis by microwave-assisted acid pyrolysis (MAP) (Chen et al., 2014) and were conjugated with CDs (Figure 1). Briefly, the lyophilized COLII was vortexed with 25% Trifluoroacetic acid (TFA) + 20 mM Dithiothreitol (DTT) for 5 m to obtain a homogeneous solution. The solution was transferred to 1.5 mL polypropylene vials and sealed with Teflon tape for processing using a domestic microwave oven (900 W, 2,450 MHz) for 3, 5, 7, and 10 m. A beaker containing 100 mL water was also placed in microwave oven during the irradiation. After microwave processing, the solutions were dried in SpeedVac for 30 m for acid removal, dissolved with 0.1% TFA, desalted in Amicon filter (cut off. 3 kDa, Merck, USA), and dried again in the SpeedVac.

After spotting of the dried samples on to the Anchor chip target plate (600/384 F, Bruker Daltonics), the spots were dried for 30 m and analyzed for MALDI-TOF by using Ultraflextreme mass spectrometer (Bruker Daltonics, Germany). Different areas of sample spot on MALDI target plate were selected for data acquisition.

The isolated peptides were conjugated with CDs by NHS/EDC coupling method. Briefly, CDs (1 mg) was diluted with 1 ml diH₂O and pH was adjusted to 5.4. For synthesis of PCDs by time-based MAP followed by NHS/EDC coupling, 1:5:5 ratio of CDs, NHS and EDC was observed to be optimal for efficient conjugation, since EDC with NHS forms an active ester with -COOH groups present on CDs surface (Bartczak and Kanaras, 2011). The NHS/EDC was added to the diluted CDs solution followed by continuous magnetic stirring at 37°C for 30 m. The dried peptides (1 mg) were introduced into the solution and magnetically stirred for 10 h followed by incubation at 4°C to stop the reaction. The peptide-conjugated CDs were dialyzed (Dialysis membrane, 3.5 kDa molecular cut-off) against diH₂O for 12 h and lyophilized.

Dynamic light scattering (DLS) and Zeta potential were recorded with a Malvern Zetasizer Nano-equipment with a temperature-controlled sample chamber by employing a 4.0 mW He−Ne laser operated at 632.8 nm and 173° angle. The raw data were processed to obtain the hydrodynamic diameter (D_h) and size distribution in terms of polydispersity index (PDI). UV-vis absorption spectrum of PCDs was performed on UV-visible spectrophotometer (Shimadzu UV-2450) and the 300–600 nm spectrum was analyzed. Further, Photoluminescence (PL) spectra were measured on a Hitachi F-4600 fluorometer equipped with Xe lamp at ambient conditions. Transmission electron microscopy (TEM, FEI Tecnai G2 F30) was performed for morphological analysis of PCDs. Attenuated Total Reflection Fourier Transform Infrared Spectroscopy (ATR-FTIR) of PCDs was performed over 4,000–700 cm^-1 range using Spectrophotometer (Thermo Nicolet NEXUS-870). Finally, nano-morphology of PCDs was analyzed by atomic force microscopy (AFM, Bruker Dimension Icon).

Capra adipose tissue-derived mesenchymal stem cells (ADMSCs) were used for the assessment of cytotoxicity and cyto-compatibility of PCDs at 1, 3, and 5 d post-incubation. Necessary approval for the isolation of ADMSCs was obtained from the Committee for Stem Cell Research (CSCR) of Indian Institute of Engineering Science and Technology (Shibpur), India vide approval no. CSCR/Decisions/3/15. The isolation and characterization of ADMSCs from Capra have been detailed in our previous work (Maity et al., 2019). Cytotoxicity of PCDs was assessed by MTT assay as per the protocol (Maity et al., 2022). Briefly, freshly prepared MTT solution (40 μL, 5 mg/mL) was added to the cells and incubated for 4 h. After carefully removing the supernatant, the formazan crystals were dissolved with DMSO and absorbance was measured using microplate reader (Bio-Rad 680, USA) at 570 nm. Live-dead assay (Invitrogen) was performed to assess viability of ADMSCs in presence of PCDs according to the protocol (Roy et al., 2019). Briefly, 1, 3 and 5 d post-incubation, PCDs and tissue culture plate (TCP) as control were washed with PBS followed by incubation with live–dead staining solution at 37°C for 30 m. After incubation, the cells were washed with PBS and observed under fluorescent microscope (Carl Zeiss) with excitation filters of 450–490 nm (green, Calcine AM) and 510–560 nm (red, ETD-1). Live-dead assay was also performed with MG63 cells for both CD and PCD-containing media after 1 and 5 days culture.

For internalization of PCDs in cells cytoplasm and nucleus, ADMSCs (1 × 10⁴) were seeded into each well of six-well plates and incubated for 24 h at 37 °C. After removal of culture media, the plates were washed with PBS followed by addition of 20 μL of PCDs (100 μg/mL) to each well at 3, 6, 12, 24, and 48 h. After incubation, the cells were visualized under confocal microscope (TCS SP8, Leica).

After trypsinization, ADMSCs were plated into six-well plates. At 70% confluence, DMEM media containing PCDs was added to the cells and incubated at 37°C in CO₂ incubator for generation of cell clusters. The clusters were transferred to pluronic F 127 hydrogel for long time culture in 3D environment for spheroid formation. For this purpose, lyophilized PCD (0.1 mg/ml) was mixed with F 127 (20% w/v) containing DMEM media to form pre-gel mix. The blend was mixed vigorously and transferred to a refrigerator for micelles formation. After complete dissolution, the solution was layered in 24 well plate followed by 1 h incubation at 37°C in CO₂ incubator for complete gelation. Cell clusters were placed on the surface of hydrogel and incubated at 37°C in CO₂ incubator for 30 m. Pre-warmed DMEM media containing PCDs was added to the system for 21 d study, while the PCDs/media was replaced every 3rd day. RNA isolation, CDNA synthesis and qPCR of cartilage specific gene amplification with GAPDH normalization was performed as per our previously published literature (Kapat et al., 2018; Mukherjee, Dhara and Saha, 2021) with the primers mentioned in appended Supplementary Table SA1.

Quantitative analysis of sulfated glycosaminoglycan (sGAG) was carried out by Alcian blue assay for evaluating the extent of chondrogenesis induced by the PCDs. For this assay, the spheroids were digested for 12 h with cysteine hydrochloride (10 mM, Sigma, USA), papain (125 μg/ml, Sigma, USA) and Na₂EDTA (2 mM, Sigma, USA) in a phosphate buffer solution (0.1 M, pH 6.8) at 60°C for 12 h. The digested solution was centrifuged at 15,000 g for 20 min to collect the supernatant. A 10% solution of Alcian Blue dye stock (1 g Alcian Blue 8GS dye in 100 ml of 18 mM H₂SO₄) was in 0.25% Triton X-100, 0.018 M H₂SO₄ was prepared, and guanidine solutions (8 M in 0.027 M H₂SO₄ and 0.375% Triton X-100) were also prepared. The Alcian blue solution was mixed with the previously collected supernatant and vortexed for 5 m followed by centrifuging at 16,000 g for 10 m at 4°C. The obtained pellets were dissolved in 500 µl of 8 M guanidine HCl by vigorous vortexing followed by centrifugation at 16,000 g for 3 m. Absorbance of the Alcian blue-sGAGs complex was measured at 595 nm wavelength using microplate reader (iMarkt, India) and the quantities of sGAGs contents were estimated by comparing the obtained data with a standard curve.

New Zealand white rabbits (n = 4) of age 12 months and weighing 2.5–3 kg were procured from Chakraborty Enterprise (Kolkata, India) and were used for the animal study. Necessary animal ethical clearance was obtained from Institutional Ethical Committee of Indian Institute Technology, Kharagpur (IIT Kharagpur), India (IE-4/SD-SMST/2.15). The animals were acclimatized for 2 weeks in the animal care facility with adequate diet prior to the surgery. For the surgical procedure, acclimatized rabbits were anesthetized by Midazolam (0.5 ml) followed by local anesthesia (Lignocaine) of the periphery of surgical zone. After sterilization by β-Iodine, cartilage defects (8 mm) were made near the center of each auricle without damaging the outer skin using a drilling machine with a drill bit of 8 mm dia. Details of the surgical procedure can be found one of our previous works (Maity et al., 2022). PCD-F127 hydrogels were injected into the defects in both right and left ears (n = 8 ears) and subsequently covered with leucoplast over a thin layer of cotton gauge. For post-operative pain management, the rabbits were treated with Ibuprofen (1ml/day) and Calmpose (0.25ml/day) for 3 days under the guidance of a veterinary surgeon.

To evaluate cartilage regeneration, the rabbits were euthanized after 15 and 60 d post-surgery with an overdose of Midazolam injection and the tissue specimens were collected and fixed in 4% v/v formalin. Fixed tissue specimens were dehydrated by treating with graded series of ethanol (70–100%) followed by tissue cleaning with xylene and finally embedded in paraffin for histological sectioning. The obtained sections from paraffin blocks were stained with hematoxylin and eosin (H&E) and Masson’s trichrome (MT, Sigma-Aldrich, USA) to assess cartilage regeneration. The stained histological samples were visualized and captured for images using an Axio Observer Z1 microscope (Carl Zeiss, Germany).

All the experiments were performed in triplicate. The results are reported as mean ± standard deviation (SD) and statistical analysis was carried out by performing one-way ANOVA using Origin 9.1 software (USA). p < 0.05 was considered statistically significant.

It was observed that both the hydrodynamic diameter (Figure 2A) and zeta potential (Figure 2B) of synthesized CDs depends upon the concentration of HNO₃. The optimum concentration of HNO₃ was found to be 50 µg/20 mL water for synthesizing zwitterion CDs with 3.98 nm average diameter and zeta potential of −0.119 mV. At low acid concentration of 25 µg/20 mL water the average diameter was higher and the zeta potential was lower, while both of these features displayed gradually increasing trends at higher concentrations.

It can be assumed that low concentration of HNO₃ resulted in inefficient pyrolysis, evidenced by larger hydrodynamic diameters and lower zeta potential. On the other hand, high acid concentration can induce negatively charged -OH, -COOH, -NO₂ functional groups on the surface of the CDs (Ray et al., 2009).

In microwave-assisted acid pyrolysis (MAP) for peptide isolation, the yield was found to be highest for 5 min of irradiation (Figure 2C), and longer exposure caused disintegration as evident from the MALDI-TOF MS analysis. Such observations are consistent with existing literature (Reiz and Li, 2010).

Peptide conjugation on the surface of CDs by NHS/EDC coupling for 24 h was optimum to generate sufficient yield of PCD. To identify the structural and functional characteristics in PCDs, several non-destructive testing like FTIR, UV, PL, AFM, and TEM were performed. Hydroxyl and other polar functional groups (-C=O, -COOH, -C-O-C-) generated through the single step top-down oxidative coupling on PCDs surface were detected through FTIR analysis (Figure 3A). FTIR spectra showed that the combined pyrolysis and carbonization process introduced oxygen-containing functional groups at 1,010–1,106 cm^-1 for C–O–C, 1,240 cm^-1 for C–OH and 1720 cm^-1 for C=O, respectively, to the edges and basal plane of PCDs. The characteristic peaks at ∼3,280 cm⁻¹ and 1,535–1,640 cm^-1 were assigned to -OH and amide functionalities, respectively. Other peaks at 810, 1,012, and 1,364 cm^-1 correspond to the presence of C-H bending and C-O stretching frequencies arising from the aromatic amino acids. These polar groups contributed to an increased water solubility of the synthesized PCDs. Further, the peaks at 1,610–1,650 cm^-1 indicate the presence of sp² hybridized honeycomb lattice (Strauss et al. 2014).

The influence of surface polarity over optical properties of PCDs was assessed by UV-vis spectroscopy, as shown in Figure 3B The absorption peaks at 258 and 460 nm with a tail extending to visible range were attributed to π–π* transition of -C=C bonds and n–π* transition of C=N bonds, respectively. The peak at 258 nm was related to the absorption of aromatic system with sp² carbon network and peak at 460 nm was attributed to carbonyl group present on the PCDs surface (Jung, Shin and Kim, 2015). It was evident from the fluorescence spectra that the PCDs showed different excitation wavelength (Figures 3C, D). The excitation wavelength of PCDs varied from 380 to 640 nm and emission spectra shifted to longer wavelengths. The maximum emission peak was found at 620 nm for excitation at 520 nm.

The TEM and AFM images in Figures 3E, F shows quasi-spherical dot-like morphology of PCDs in the size range 2–8 nm. The size distribution obtained through DLS study was calculated in the range of 2.5–8.0 nm, which is little bit higher than the data obtained from TEM analysis. Due to high surface polarity of the PCDs, they have easy solvation in protic media.

MTT assay (Figure 4A) established that the cell populations with PCDs at 1, 3, and 5 d were significantly higher than those with CDs (p ≤ 0.05). Cell population almost doubled on day 5 from day 3. Live-dead assay (Figures 4B–D) also showed relatively higher proliferation of cells with cluster formation on 5^th day as compared to 3 d of culture. These results confirmed the cytocompatibility of PCDs, and indicated that the PCDs could be internalized by cell nuclei and could modulate the cell behavior to form clusters. Results of the Live-dead assay for MG63 cells are appended as Supplementary Figure SA1.

During confocal imaging, excitation of PCDs with 365, 488, and 580 nm wavelengths produced blue, green and red fluorescence, respectively. The fluorescence of PCDs started to appear in cell cytoplasm at 30 m and subsequently migrated to nucleus within 24 h of incubation. The highest localization of PCDs in nucleus was obtained at 48 h (Figure 5). Neutrally charged carbon nano-dots with size less than 9 nm is known to be transported to nuclei within 24 h of seeding (Jung, Shin and Kim, 2015), and therefore the multicolor fluorescent PCDs can be effective in non-invasive monitoring of tissue in-growth and healing of wound.

Within 24 h of seeding, the ADMSCs moved closer to each other (Figure 6A), towards formation of clusters at day 3 (Figure 6B). Post 5 d culture, almost all the cells migrated to those clusters (Figure 6C).

In the hydrogel culture, the clusters matured into spheroids, and were evaluated for cartilage related gene (COLII, SOX 9 and aggrecan) expression using qRT-PCR analysis. In spheroid culture, expression of COLII and aggrecan (ACAN) was upregulated and SOX 9 was downregulated as compared to control at 21 d study (Figure 6D), pointing towards the potential of peptide-conjugated carbon nano-dots to stimulate cellular differentiation and tissue formation. The results of the Alcian Blue assay (Figure 6E) also suggest presence of a significantly higher quantity of sGAG in the cells cultured in the media containing peptide-conjugated carbon nano-dots (PCDs) as compared to the cells in the native carbon nano-dots (CDs) containing media, evidencing the strong chondro-inductive abilities of the synthesized PCDs.

Gross observation of auricular cartilage defect: There were no apparent signs of infection or graft rejection at the defect region. Thicker skin tissue formation in PCDs group was observable as compared to the control groups 15 d post-surgery (Figure 7A). On 60 d evaluation, presence of dense cartilage tissue could be identified in defect area of PCDs group whereas thick skin remained in the control group (Figure 7B), without any cartilage tissue.

Histological analysis: H&E-stained sections of retrieved specimens at 15 and 60 d revealed that the defect region of auricular cartilage in the control area did not show any presence of chondrocyte at both 15 and 60 d post-surgery (Figures 7C, D), and the treatment area was also almost devoid of chondrocytes 15 d post-implantation of PCDs-laden hydrogel (Figure 7E), but the specimens taken 60 d post-implantation evidenced significant regeneration of cartilage tissue with strong presence of chondrocytes (Figure 7F).

The histochemical analysis of cartilage regeneration in the defect area is presented in Figure 8. Since the Alcian blue stain binds strongly to sulfated GAGs and glycoproteins, and cartilage tissue contains the highest concentrations of GAGs than any other tissue, newly-formed cartilage appears blue in the MT-stained tissue samples. No significant tissue growth could be observed in the control group, both 15 d and 60 d post-implantation (Figures 8A, D, respectively). No capsular layer was observable in any of the tissues, indicating healthy integration of newly-formed tissues with existing ones and absence of fibrosis. 15 d study of the treated zone showed growth of neo-cartilage with a reduced defect size (Figures 8C, D), whereas dense cartilage with structurally formed lacuna was prominent in the treated defect 60 d post-implantation (Figures 8E, F).