Osteochondral Defects Healing Using Extracellular

Article Cite This: ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

Osteochondral Defects Healing Using Extracellular Matrix Mimetic Phosphate/Sulfate Decorated GAGs-Agarose Gel and Quantitative Micro-CT Evaluation Kausik Kapat,*,† Arun Prabhu Rameshbabu,† Priti Prasanna Maity,‡ Abhisek Mandal,§ Kamakshi Bankoti,† Joy Dutta,† Deb Kumar Das,† Goutam Dey,† Mahitosh Mandal,† and Santanu Dhara*,† †

School of Medical Science & Technology, and §Department of Chemistry, Indian Institute of Technology Kharagpur, Kharagpur 721302, India ‡ Centre for Healthcare Science and Technology, Indian Institute of Engineering Science and Technology, Shibpur 711103, India S Supporting Information *

ABSTRACT: Tissue engineering has a major emphasis in creating tissue specific extracellular ambiance by altering chemical functionalities of scaffold materials. Heterogeneity of osteochondral tissue necessitates tailorable bone and cartilage specific extracellular environment. Carboxylate- and sulfate-functionalized glycosaminoglycans (GAGs) in cartilage extracellular matrix (ECM) create an acidic ambience to support chondrogenic activity, whereas phosphaterich environment in bone enables chelation of calcium leading to the formation of mineralized matrix along with an alkaline environment to support osteogenesis. In this study, chitosan, a naturally occurring GAGs, was functionalized with phosphate/sulfate groups analogous to bone/cartilage ECM and incorporated in thermogelling agarose hydrogel for delivery to osteochondral defects. In vitro studies revealed significantly higher adhesion and proliferation of adipose derived mesenchymal stem cells (ADMSCs) with blended hydrogels as compared to that of native agarose. Cell differentiation and RT-PCR studies of the phosphorylated hydrogels revealed higher osteogenic potential, while sulfated hydrogels demonstrated enhanced chondrogenic activity in comparison to agarose. Recovery of osteochondral defects after delivery of the thermoresponsive agarose-based hydrogels decorated with phosphorylated derivatives showed significantly higher bone formation. On the other hand, cartilage formation was significant with chitosan sulfate decorated hydrogels. The study highlights the role of chitosan derivatives in osteochondral defect healing, especially phosphorylated ones as bone promoter, whereas sulfated ones act as cartilage enhancer, which was quantitatively distinguished through micro-CT-based noninvasive imaging and analysis. KEYWORDS: osteochondral defects, extracellular matrix (ECM), glycosaminoglycans (GAGs), chitosan sulfate, chitosan phosphate, thermoresponsive hydrogel

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INTRODUCTION In recent times, osteochondral defects (OCD) have been common because of higher life expectancy and an increase in elderly population. Traumatic injury, repetitive microtrauma, and other age-related factors are often responsible for damage in the osteochondral region, leading to severe joint pain, functional disability, and reduced quality of life. Osteochondral defects healing is usually a slow process due to inadequate supply of reparative cells and biochemical factors in avascular cartilage.1 Among the available techniques, microfracture is commonly preferred for small osteochondral defects ( 0.05) in AGCP as compared to AGCS and AG, whereas AGCS exhibited significantly higher (p < 0.05) expression than that of AG. Interestingly, expression of collagen I, the bone-specific extracellular protein marker, was detected after 21 d by immunostaining (Figure 10c), which also revealed comparatively higher expression of collagen I in AGCP scaffolds as compared to the other samples in this study. Chondrogenic Differentiation and Gene Expression Analysis. Chondrogenic potential of the hydrogel scaffolds was evaluated through gene (COL II, ACAN and SOX9) expression analysis and compared after 21 d of ADMSCs culture using chondrogenic supplementation. Corresponding RT-PCR profile as well as relative band intensities of chondrogenic genes expressions is shown in Figure 11a, b, respectively. In chondrogenic differentiation studies, COL II gene expression was significantly higher (p < 0.001) in AGCS when compared with AGCP and AG, but AGCP could not show significantly higher (p > 0.05) expression in comparison to AG. Other chondrogenic related genes, ACAN and SOX9, were expressed in significantly higher amount (p < 0.001 and p < 0.05, respectively) in both AGCP and AGCS as compared to AG (Figure 11b). Interestingly, ACAN and SOX9 gene expression was significantly higher (p < 0.001) in AGCS than that of AGCP. Therefore, AGCS would promote more cartilogenesis than both AG and AGCP. Herein, collagen II extracellular cartilage-specific protein marker, was expressed more after 21 d (Figure 11c) in AGCS as compared to AGCP and AG scaffolds supporting the RT-PCR data. CAM Assay. The capacity of the hydrogels to induce blood vessel formation was quantitatively estimated through CAM assay, as shown in Figure 12. After 3 d of implantation, only few chorioallantoic vessels (6 ± 2 nos.) were formed at the bottom of the translucent AG

Table 3. Roughness and Mechanical Properties of AG, AGCP, and AGCS Hydrogel roughness (AFM)a samples

Rq (nm)

Ra (nm)

Young’s modulus (MPa)

stiffness (pN/ nm)

AG AGCP AGCS

7.6 ± 0.8 8.1 ± 1.5 6.2 ± 1.3

5.7 ± 0.7 6.1 ± 1.5 4.3 ± 0.7

198.3 ± 18.2 147.6 ± 33.7 135.6 ± 22.1

614.0 ± 33.1 566.6 ± 27.6 483.6 ± 41.6

a

Rq, root mean square (RMS); Ra, average roughness

of culture as indicated by the green color of the cells after staining. The absence of red color in all images indicates nil or minimum cell death occurred on these scaffolds. Also, cells were uniformly distributed throughout the hydrogels. Further, cell morphology was analyzed by rhodamine-phalloidin/DAPI am staining. As shown in Figure 9II, most of the adhered cells were elongated while possessing typical morphology of the stem cells. However, AGCP and AGCS hydrogels exhibited higher cell density along with more cell−cell contact as compared to AG. Analysis of total DNA content at different time points provides an approximation of the change in the total cell population. Here, total DNA contents increased for all hydrogels cultured with ADMSCs which indicates continuous cell growth from day 1 to day 7. After normalization with a total weight of hydrogels, there was an increase of total DNA contents more than 2-folds in case of AGCP and AGCS hydrogels, whereas AG exhibited an increase of about 1.7-folds. The results indicate that cell population in AGCP and AGCS hydrogels was almost doubled after 7 d of ADMSCs culture. Osteogenic Differentiation and Gene Expression Analysis. Osteogenic potential of AGCP and AGCS hydrogels with respect to AG hydrogel was evaluated after 21 d of osteogenic culture using ADMSCs. Expression of osteogenic related genes (COL I, OPN, and OCN) on individual scaffolds was evaluated through RT-PCR study. RT-PCR profile and relative expressions of genes assessed from different band intensities are shown in Figure 10a, b, respectively. H

DOI: 10.1021/acsbiomaterials.8b00253 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering

Figure 7. (a−c) AFM micrographs and (a′−c′) force versus indentation displacement curves obtained after analyzing lyophilized AG, AGCP, and AGCS scaffolds, respectively.

Figure 8. (a) Swelling and (b) degradation studies of lyophilized scaffolds (a) AG, (b) AGCP, and (c) AGCS.

scaffolds along with a few approaching blood vessels (9 ± 3 nos.) toward the scaffolds. For AGCP scaffolds, there was an insignificant change in the number of blood vessels (6 ± 3 nos.) as compared to AG. However, significantly higher number (18 ± 3 nos.) of approaching blood vessels converged toward the scaffolds were found in this case. In contrast, AG-CS exhibited insignificant counts of blood vessels at the bottom (4 ± 2 nos.) as well as surrounding region (5 ± 2 nos.) of the scaffolds. The obtained results confirmed that the converging blood vessels toward AGCP were significantly (p < 0.001) higher than that of AGCS and AG indicating the angiogenic potential of CP. Animal Study. After 12 weeks of healing in the presence of different hydrogels, the bone specimens were retrieved and optical images were captured for macroscopic evaluation of the

defects region (Figure 13a). Unperturbed healing was observed in all cases without any major sign of infection or inflammation. Reddish tissues were formed in the defect region with distinctly identifiable defect boundaries (marked by red dotted circles) after 12 weeks of healing. Although, defects filled with AGCS and AGCP hydrogels were healed properly, partial healing of the defect implanted with AG hydrogel was also observed. Micro-CT Evaluation. Qualitative and quantitative healing of the osteochondral defects were evaluated by micro-CT after 12 weeks and the effects of various hydrogels on bone ingrowth were compared. Retrieved bone specimens were scanned at the region of interest (ROI) and percentages of bone volume (BV) to total defect volume (TV) were calculated for each defect region using VGStudio MAX. Three dimensional views from the I


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Figure 9. Assessment of in vitro cytotoxicity and cell morphology through (I) live−dead (green color, live; red color, dead cells) and (II) rhodaminephalloidin/DAPI staining (red color, cytoskeleton; blue color, nucleus) on 5d; (III) cell proliferation through DNA quantification on 1d and 7d using ADMSCs seeded (a) AG (b) AGCP (c) AGCS hydrogels.

Figure 10. Differentiation of ADMSCs after 21 d of culture on AG, AGCP and AGCS under osteogenic supplementation (a) RT-PCR profile, (b) relative band intensities, and (c) immunostaining of secreted collagen I in the extracellular matrix (green and blue color represent extracellular collagen and cell nucleus, respectively). Error bar represents standard deviation (n = 3).

Figure 11. Differentiation of ADMSCs after 21 d of culture on AG, AGCP, and AGCS under chondrogenic supplementation: (a) RT-PCR profile, (b) relative band intensities, and (c) immunostaining of secreted collagen II in the extracellular matrix (green and blue color represent extracellular collagen and cell nucleus, respectively). Error bar represents standard deviation (n = 3).

higher bone ingrowth as compared to AG hydrogel (Figure 13f). BV/TV estimated for AG, AGCP and AGCS implanted region were 31.7, 53.0, and 48.5%, respectively (Figure 13h). However, none of the 3D images clearly showed the actual state of cartilage regeneration.

top and cross-section of retrieved bone specimen are represented in Figure 13b, c, respectively. Figure 13d, e represent side and top views of cylindrical regions, respectively extracted from defects using VGStudio MAX software and show differential healing of defects containing AGCS and AGCP hydrogels with relatively J


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Figure 12. Chick chorioallantoic membrane (CAM) assay using lyophilized hydrogel scaffolds: (a) AG, (b) AGCP, and (c) AGCS; (d) plots indicating blood vessels counts below and surrounding the scaffolds. Error bar represents standard deviation (n = 3).

Figure 13. Osteochondral healing in rabbit model after 12 weeks of AGCS, AGCP, and AG hydrogels implantation: (a) macroscopic view; micro-CT images (b) top and (c) cross-sectional view; (d) side view (defect regions are marked with dotted circles), (e) top view, and (f) 3D view of extracted bone from defects regions by VGStudio MAX; (g) bone and cartilage regions in 2D micro-CT slices using MATLAB (blue, cyan, and yellow color represents cartilage, bone, and the background, respectively); (h) bone volume with respect to total defects volume obtained from 3D micro-CT images; and (i) bone and cartilage area percentages with respect to total defect area obtained from 2D micro-CT slices.

Bone and cartilage areas from 2D micro-CT slices (n = 5) were extracted by using Otsu thresholding approach, where blue regions represent cartilage, cyan color represents bone area, and yellow represents background, respectively (Figure 13g). It is to be noted that top yellow region was excluded from total void area calculation since this was part of the sample. Bone, cartilage and

void in the tissue region were considered for calculation of individual as well as total area based on the number of pixel values. As plotted in Figure 13i, percentages of bone area (BA) with respect to total area (TA) were calculated to be 45.8 ± 4.2, 60.0 ± 4.5 and 52.5 ± 0.8, whereas percentages of cartilage area (CA) with respect to total area (TA) were 6.0 ± 0.5, 5.9 ± 0.7 K


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ACS Biomaterials Science & Engineering and 10.7 ± 0.6, for defects filled with AG, AGCP and AGCS, respectively. The statistical analysis clearly indicates that the quantified value does not reject the null hypothesis at 5% significance level.

MW of the functionalized polymers was observed due to breakdown of native chitosan backbone during modification reaction at high acidic pH and temperature. This significantly affected the pore characteristics and other physical properties of the lyophilized hydrogel scaffolds prepared after blending with AG (MW ∼ 1.2 × 105 Da). Because of the higher MW, chain entanglement was at a maximum with CP, followed by CS and AG. On the other hand, H-bonding was maximum with AG, followed by CP and CS, based on availability of free −OH groups. Sublimation of ice crystals during lyophilization resulted in a much smaller pore size of AGCP due to limited mobility of polymer chains. Swelling of hydrogel plays important role during cellular migration. The extent of swelling was observed in the order of AGCS > AGCP > AG, which was mainly due to the differential swelling properties of chitosan derivatives depending upon their MW and chain entanglement. It is to be noted that the extent of chain entanglement is directly proportional to MW of the linear chain polymers. Here, viscosity average molecular weights (Mv) of phosphate and sulfate functionalized chitosan derivatives were estimated to be 3.1 × 105 Da and 2.2 × 105 Da, respectively and degree of chain entanglement in AGCP was comparatively more than AGCS scaffolds due to higher MW of CP. On the other hand, extensive H-bonding in AG facilitated formation of a stable hydrogel network that allowed less swelling of AG scaffolds in PBS (pH 7.4) at 37 °C. High swelling index signifies high fluid retention ensuring supply of essential nutrients for cellular growth. As soon as the material swells, pore diameter also increases, which not only facilitates cell attachment but also allows cells to penetrate to the bulk.63,64 Biodegradation is an important property of the biomaterials. Ideally, scaffolds should degrade at a similar rate to neotissue formation.65 AG lacks sufficient biodegradability because of the absence of specific enzymes in a mammalian system, but CH does not have such issues. Therefore, blended AGCP and AGCS hydrogels degraded relatively faster than that of AG hydrogels. Besides enzymatic degradation of glycoside linkages in CH derivatives, faster degradation of blended hydrogel was also due to polymer dissolution in absence of chemical cross-linking. Scaffold morphology plays a significant role in differentiation of cells. Although, there have been conflicting reports on the optimum pore size of the polymeric scaffolds for the highest chondrogenic and osteogenic differentiation. Stenhamre et al.66 reported that pore size 150 μm. Oh et al.67 demonstrated that 370−400 μm pore size is optimum for chondrogenic differentiation of adipose stem cells. According to Matsiko et al.,68 300 μm pore size stimulates higher chondrogenic gene expression and cartilage-like matrix production as compared to scaffolds with smaller pores (94 and 130 μm). Other studies involving CH or gelatin scaffolds also exhibited improved chondrocyte activity using 250−500 μm pore size.69,70 In this study, AGCP and AGCS hydrogels exhibited higher cell density and more cell−cell contact as compared to AG hydrogels, since CH has more cell adhesion property than AG.71 Estimation of total DNA content supported similar observations as blended hydrogels exhibited higher DNA contents in comparison to native AG scaffolds. There was continuous cell growth on all three hydrogels as indicated by an increasing order of total DNA from day 1 to day 7 of cell culture. Expressions of COL I and OPN (osteogenic related) genes were significantly higher for the

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DISCUSSION GAGs in articular cartilage are usually present in the form of proteoglycans that constitute 10 wt % of the total wet mass of cartilage. Aggrecan is the most abundant proteoglycan that contains chondroitin sulfate (80%), keratan sulfate (5−20%) and hyaluronan (1−10%).58 Smaller percentages of GAGs together with aggrecan, collagen and other link proteins form a stable viscoelastic hydrogel framework that contributes to the loadbearing properties of articular cartilage. On the other hand, proteinaceous matrix in native bone contributes to 10−30% of its wet weight, in which 1/10th mass is constituted by noncollagenous proteins (NCPs).59 NCPs are SIBLING (small integrin-binding ligand, N-linked glycoprotein) group of proteins that consist of five key members− osteopontin (OPN), matrix extracellular phosphoglycoprotein (MEPE), bone sialoprotein (BSP), dentin matrix protein 1 (DMP1), and dentin sialophosphoprotein (DSPP). Although NCPs including phosphoproteins are present in minor quantity (1−2 wt %), they play vital role in regulating cellular adhesion, bone homeostasis and bone mineralization. In this study, sulfated GAGs and phosphoprotein mimetic sulfate/phosphate functionalized hydrogels were prepared from low concentrations (2 wt %) of CS or CP solutions blended with AG solution (2 wt %) in equal volume, in order to mimic similar percentages of sulfated GAGs and phosphoproteins occurring in bone and cartilage, respectively. The thermogelling AG hydrogel was used as a carrier for delivering CP and CS into the osteochondral defects. Although, AG is considered as gold standard material for cartilage tissue engineering, slow degradability and poor cell adhesion properties60 restrict its in vivo application to some extent. Blending of AG with other biodegradable polymers modulates its functional properties.61,62 No additional cross-linking step was incorporated for preparing hydrogels to avoid toxic interferences. The preformed hydrogels were compared with each other regarding physicochemical and mechanical properties. There was decrease in gel strength in blended hydrogels as compared to native AG hydrogel. Higher gel strength of AG was attributed to extensive intermolecular H-bonding and chain entanglement. Besides insignificant ionic interactions between neutral AG molecule with negatively charged CP and CS at physiological pH, a significant reduction in gel strength was mainly due to a lesser extent of chain entanglement as a result of molecular weight degradation during functionalization. All hydrogels exhibited strain reversibility property and therefore they are able to undergo repetitive loading cycles especially at articular cartilage site without plastic deformation. Porosity more than 80% with high pore interconnectivity was obtained for the hydrogels through micro-CT analysis. High pore volume fraction along with pore interconnectivity is essential for nutrients transport and cellular migration. With significant reduction in total porosity (83.3% ± 1.4%), the average pore diameter reduced significantly in AGCP (69.6 ± 36.6 μm) as compared to that of AG (124.1 ± 62.4 μm) and AGCS (117.6 ± 79.5 μm) with 92 ± 1% and 95.6 ± 1.5% total porosities, respectively. In this study, hydrogel network was formed through physical interactions, such as chain entanglements and Hbonding, in absence of any chemical cross-linking. A decrease in L


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ACS Biomaterials Science & Engineering AGCP and AGCS hydrogels as compared to that of AG. More specifically, cells on AGCP expressed more COL I genes than that of AGCS. This indicates CP has higher osteogenic potential than CS. However, difference in OCN related genes expression in all the scaffolds was insignificant, since OCN is usually expressed more until 14 d and subsequently decreases up to 21 d. Chondrogenic related genes, COL II, ACAN, and SOX9, were expressed with significantly higher intensity for AGCS scaffolds as compared to AG and AGCP. AGCP hydrogel blend did not show any significant difference in chondrogenic related gene expression compared to AG, since AG is already known for its chondrogenic ability. AG is considered to be a gold standard biomaterial and superior base for long-term chondrocyte culture, maintenance of regular cartilage specific genes (aggrecan and collagen II) expression and higher GAG deposition in the ECM.72−74 Because of the sulfated GAG mimetic environment in AGCS, chondrogenic related genes were expressed comparatively more than the other scaffolds. This signifies that although CP and CS are able to induce both osteogenic and chondrogenic differentiation of ADMSCs, CP induces more osteogenic differentiation, whereas CS has higher capacity to induce chondrogenic differentiation. These findings were further supported by immunostaining of extracellular collagen, where of collagen I was secreted more from the cells seeded on AGCP scaffolds as compared to AGCS with more collagen II secretion. None of the hydrogels adversely affected chorioallantoic vessels formation, as evident from the blood vessel counts at the bottom of the scaffolds. However, approaching blood vessels increased significantly toward AGCP as compared to the other scaffolds. In AGCS, blood vessel counts were decreased than control. This study signifies that AGCP has proactive role in neovascularization. To support high metabolic activity in bone, adequate vascularization is essential. Rapid gaseous and nutritional transport, removal of metabolic byproducts and delivery of hormones, growth factors etc. are carried out in bone through blood supply. Bone graft substitute materials should support neovasculaturization for accelerated bone regeneration. In contrary, since cartilage is avascular with limited tissue regeneration, lesser nutritional demand for low chondrocytes population could be fulfilled only through diffusional transport. For chondrogenic activity without matrix mineralization, absence of vasculature along with hypoxic environment is always preferable. Although CAM assay is suitable only for short-term angiogenesis study, long-term experiment is necessary for ensuring blood vessels penetration into the scaffolds through subcutaneous implantation. Healing efficacy of phosphate/sulfate functionalized AG hydrogels was evaluated in rabbit osteochondral defects model. Although larger animals, like monkeys, dogs, sheeps, pigs, goats, horses etc., are usually preferred for study of osteochondral defects due to anatomical resemblance of articular cartilage structure to that of human, high experimental cost is the major constraint for involving such models. Osteochondral defects in rabbits were created as 6 mm diameter × 6 mm depth. Implantation of functionalized hydrogels through in situ gelation was found to be advantageous, because this overrules the customization step. Also, functionalized hydrogel delivery was carried out on the basis of a cell-free approach without the necessity of gluing, which is essential for scaffold preforms at the defect site. The porous nature of such injectable hydrogels allows infiltration, proliferation and differentiation of bone marrow stromal cells, as already demonstrated through in vitro ADMSCs culture in the present work. Previous studies by Nagura et al.75

reported improved cartilage healing using poly(DL-lactide-coglycolide) (PLG) based cell-free scaffolds. They also reported significant cartilage healing in 24 weeks, whereas present study showed similar extent of osteochondral defect healing in 12 weeks while considering 5 mm depth and 5 mm diameter defect size. Gupta et al.76 reported healing of relatively smaller subchondral defects (4 mm depth and 4 mm diameter) using chitosan-agarose-gelatin (CAG) based cryogel scaffolds, where regenerated cartilage had structural similarity with native one in 8 weeks. Further, the study utilized custom-made scaffolds along with surgical glue as fixation aid to immobilize the scaffold into the defect site. Similarly, hyaluronan-based scaffolds delivered to relatively smaller defects (3 mm depth and 3 mm diameter) exhibited healing in 12 weeks.77 However, histological studies would be required to investigate details of the newly grown tissue. In the present context, grafting of porous titanium78,79 at the osteochondral defect site revealed accelerated tissue regeneration80 as evidenced from micro-CT analysis and histological studies. Further, hydrothermally modified porous titanium samples with calcium phosphate deposited titanate nanostructures81 showed increased osteogenic response and bone ingrowth.82 Site-specific infiltration of synthetically derived phosphate/sulfate GAGs as biomimetic ECM analogue into porous titanium as template for osteochondral defects healing would an interesting proposition.

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CONCLUSIONS The study was designed to distinguish the effects of CP and CS on bone and cartilage formation; therefore hydrogels were prepared from these functional derivatives with thermogelling AG. ADMSCs differentiation studies using specific supplementation revealed comparatively higher osteogenic related gene expression on AGCP in contrast to higher expression of chondrogenic related genes on AGCS. Further, AGCP attracted more blood vessels on chick chorioallantoic membrane (CAM), whereas AGCS supported inferior vascularization as compared to AG. When delivered to the osteochondral defects in rabbit femoral bone, recovery of cartilage was significantly higher with AGCS, while AGCP exhibited accelerated bone formation. The native hydrogel of AG exhibited inferior healing of both bone and cartilage tissues. The study provides a future scope for creating phosphate/sulfate graded biphasic scaffolds, cell−material constructs or extracellular ambience through site specific infiltration of phosphate/sulfate hydrogel in porous ceramic/ metal structures for accelerated osteochondral healing. This could be further explored in the realm of tissue engineering through a biomimetic approach.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.8b00253. Additional data for changes in storage, loss, and complex modulus against frequency sweep studies on preformed hydrogels; elemental distribution, EDX mapping, and FTIR spectra of the lyophilized scaffolds (PDF)

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AUTHOR INFORMATION

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*E-mail: [email protected] (K.K.). M


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ACS Biomaterials Science & Engineering *E-mail: [email protected]. Tel.: +91-3222-282306. Fax: +91-3222-255303 (S.D.).

(15) Liu, X. J.; Feng, Q. L.; Bachhuka, A.; Vasilev, K. Surface modification by allylamine plasma polymerization promotes osteogenic differentiation of human adipose-derived stem cells. ACS Appl. Mater. Interfaces 2014, 6, 9733−9741. (16) Glennon-Alty, L.; Williams, R.; Dixon, S.; Murray, P. Induction of mesenchymal stem cell chondrogenesis by polyacrylate substrates. Acta Biomater. 2013, 9, 6041−6051. (17) Cramer, G. D.; Bakkum, B. W. Microscopic Anatomy of the Zygapophysial Joints, Intervertebral Discs, and Other Major Tissues of the Back. In Clinical Anatomy of the Spine, Spinal Cord, and ANS; Elsevier: Amsterdam, 2013; pp 586−637. (18) Herring, G. M. The chemical structure of tendon, cartilage, dentin and bone matrix. Clin. Orthop. Relat. Res. 1968, 60, 261−300. (19) Murphy, W. L.; Mooney, D. J. Bioinspired growth of crystalline carbonate apatite on biodegradable polymer substrata. J. Am. Chem. Soc. 2002, 124, 1910−1917. (20) Nuttelman, C. R.; Benoit, D. S.; Tripodi, M. C.; Anseth, K. S. The effect of ethylene glycol methacrylate phosphate in PEG hydrogels on mineralization and viability of encapsulated hMSCs. Biomaterials 2006, 27, 1377−1386. (21) Sophia Fox, A. J.; Bedi, A.; Rodeo, S. A. The basic science of articular cartilage: structure, composition, and function. Sports Health 2009, 1, 461−468. (22) Urban, J. P.; Smith, S.; Fairbank, J. C. Nutrition of the intervertebral disc. Spine 2004, 29, 2700−2709. (23) Bibby, S. R.; Jones, D. A.; Lee, R. B.; Yu, J.; Urban, J. P. The pathophysiology of the intervertebral disc. Jt., Bone, Spine 2001, 68, 537−542. (24) Razaq, S.; Wilkins, R. J.; Urban, J. P. The effect of extracellular pH on matrix turnover by cells of the bovine nucleus pulposus. European Spine Journal 2003, 12, 341−349. (25) Li, H.; Liang, C.; Tao, Y.; Zhou, X.; Li, F.; Chen, G.; Chen, Q. X. Acidic pH conditions mimicking degenerative intervertebral discs impair the survival and biological behavior of human adipose-derived mesenchymal stem cells. Exp. Biol. Med. 2012, 237, 845−852. (26) Bandyopadhyay-Ghosh, S. Bone as a collagen-hydroxyapatite composite and its repair. Trends Biomater. Artif. Organs 2008, 22, 116− 124. (27) Chai, Y. C.; Roberts, S. J.; Schrooten, J.; Luyten, F. P. Probing the osteoinductive effect of calcium phosphate by using an in vitro biomimetic model. Tissue Eng., Part A 2011, 17, 1083−1097. (28) Monfoulet, L. E.; Becquart, P.; Marchat, D.; Vandamme, K.; Bourguignon, M.; Pacard, E.; Viateau, V.; Petite, H.; LogeartAvramoglou, D. The pH in the microenvironment of human mesenchymal stem cells is a critical factor for optimal osteogenesis in tissue-engineered constructs. Tissue Eng., Part A 2014, 20, 1827−1840. (29) Galow, A. M.; Rebl, A.; Koczan, D.; Bonk, S. M.; Baumann, W.; Gimsa, J. Increased osteoblast viability at alkaline pH in vitro provides a new perspective on bone regeneration. Biochemistry and Biophysics Reports 2017, 10, 17−25. (30) Tao, S. C.; Gao, Y. S.; Zhu, H. Y.; Yin, J. H.; Chen, Y. X.; Zhang, Y. L.; Guo, S. C.; Zhang, C. Q. Decreased extracellular pH inhibits osteogenesis through proton-sensing GPR4-mediated suppression of yes-associated protein. Sci. Rep. 2016, 6, 26835. (31) Varghese, S.; Hwang, N. S.; Canver, A. C.; Theprungsirikul, P.; Lin, D. W.; Elisseeff, J. Chondroitin sulfate based niches for chondrogenic differentiation of mesenchymal stem cells. Matrix Biol. 2008, 27, 12−21. (32) Collins, M. N.; Birkinshaw, C. Hyaluronic acid based scaffolds for tissue engineeringA review. Carbohydr. Polym. 2013, 92, 1262−1279. (33) Correia, C. R.; Moreira-Teixeira, L. S.; Moroni, L.; Reis, R. L.; van Blitterswijk, C. A.; Karperien, M.; Mano, J. F. Chitosan scaffolds containing hyaluronic acid for cartilage tissue engineering. Tissue Eng., Part C 2011, 17, 717−730. (34) Kim, I. Y.; Seo, S. J.; Moon, H. S.; Yoo, M. K.; Park, I. Y.; Kim, B. C.; Cho, C. S. Chitosan and its derivatives for tissue engineering applications. Biotechnol. Adv. 2008, 26, 1−21.

ORCID

Mahitosh Mandal: 0000-0003-3861-3323 Santanu Dhara: 0000-0003-4443-7610 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was financially supported by the Defence Research and Development Organization (DRDO) (Grant DLS/81/ 48222/LSRB-241/BDB/2012), Ministry of Human Resource and Development (MHRD) (Grant IIT/SRIC/SMST/DJR/ 2013-14/224), Government of India, BIRAC-SRISTI and Department of Biotechnology (DBT) (Grant BIRAC SRISTI PMU-2016/004). The authors acknowledge the contributions of Subhodeep Jana for micro-CT characterizations. The authors also acknowledge Central Research Facility (CRF), IIT Kharagpur, for support in materials characterization.

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REFERENCES

(1) Mano, J.; Reis, R. Osteochondral defects: present situation and tissue engineering approaches. J. Tissue Eng. Regener. Med. 2007, 1, 261− 273. (2) Kreuz, P. C.; Steinwachs, M. R.; Erggelet, C.; Krause, S. J.; Konrad, G.; Uhl, M.; Südkamp, N. Results after microfracture of full-thickness chondral defects in different compartments in the knee. Osteoarthritis and Cartilage 2006, 14, 1119−1125. (3) Espregueira-Mendes, J.; Pereira, H.; Sevivas, N.; Varanda, P.; Da Silva, M. V.; Monteiro, A.; Oliveira, J. M.; Reis, R. L. Osteochondral transplantation using autografts from the upper tibio-fibular joint for the treatment of knee cartilage lesions. Knee Surgery, Sports Traumatology, Arthroscopy 2012, 20, 1136−1142. (4) Vasiliadis, H. S.; Wasiak, J. Autologous chondrocyte implantation for full thickness articular cartilage defects of the knee. Cochrane Database Syst. Rev. 2010, CD003323, DOI: 10.1002/ 14651858.CD003323.pub3 (5) Yan, L. P.; Oliveira, J. M.; Oliveira, A. L.; Reis, R. L. Current concepts and challenges in osteochondral tissue engineering and regenerative medicine. ACS Biomater. Sci. Eng. 2015, 1, 183−200. (6) Bitton, R. The economic burden of osteoarthritis. Am. J. Manag. Care 2009, 15, S230−S235. (7) O’brien, F. J. Biomaterials & scaffolds for tissue engineering. Mater. Today 2011, 14, 88−95. (8) Tibbitt, M. W.; Rodell, C. B.; Burdick, J. A.; Anseth, K. S. Progress in material design for biomedical applications. Proc. Natl. Acad. Sci. U. S. A. 2015, 112, 14444−14451. (9) Saha, K.; Pollock, J. F.; Schaffer, D. V.; Healy, K. E. Designing synthetic materials to control stem cell phenotype. Curr. Opin. Chem. Biol. 2007, 11, 381−387. (10) Anderson, D. G.; Putnam, D.; Lavik, E. B.; Mahmood, T. A.; Langer, R. Biomaterial microarrays: rapid, microscale screening of polymer−cell interaction. Biomaterials 2005, 26, 4892−4897. (11) Anderson, D. G.; Levenberg, S.; Langer, R. Nanoliter-scale synthesis of arrayed biomaterials and application to human embryonic stem cells. Nat. Biotechnol. 2004, 22, 863. (12) Flaim, C. J.; Chien, S.; Bhatia, S. N. An extracellular matrix microarray for probing cellular differentiation. Nat. Methods 2005, 2, 119. (13) Keselowsky, B. G.; Collard, D. M.; García, A. J. Surface chemistry modulates fibronectin conformation and directs integrin binding and specificity to control cell adhesion. J. Biomed. Mater. Res., Part A 2003, 66, 247−259. (14) Curran, J.; Chen, R.; Hunt, J. Controlling the phenotype and function of mesenchymal stem cells in vitro by adhesion to silanemodified clean glass surfaces. Biomaterials 2005, 26, 7057−7067. N


Article

ACS Biomaterials Science & Engineering (35) Yin, Y. J.; Luo, X. Y.; Cui, J. F.; Wang, C. Y.; Guo, X. M.; Yao, K. D. A Study on Biomineralization Behavior of N-Methylene Phosphochitosan Scaffolds. Macromol. Biosci. 2004, 4, 971−977. (36) Zhang, K.; Peschel, D.; Helm, J.; Groth, T.; Fischer, S. FT Raman investigation of novel chitosan sulfates exhibiting osteogenic capacity. Carbohydr. Polym. 2011, 83, 60−65. (37) Cao, L.; Wang, J.; Hou, J.; Xing, W.; Liu, C. Vascularization and bone regeneration in a critical sized defect using 2-N, 6-O-sulfated chitosan nanoparticles incorporating BMP-2. Biomaterials 2014, 35, 684−698. (38) Tang, T.; Zhang, G.; Lau, C. P.; Zheng, L. Z.; Xie, X. H.; Wang, X. L.; Wang, X. H.; He, K.; Patrick, Y.; Qin, L.; Kumta, S. M. Effect of watersoluble P-chitosan and S-chitosan on human primary osteoblasts and giant cell tumor of bone stromal cells. Biomed. Mater. 2011, 6, 015004. (39) Zhu, Y.; Wang, X.; Cui, F. Z.; Feng, Q. L.; De Groot, K. In vitro cytocompatibility and osteoinduction of phosphorylated chitosan with osteoblasts. J. Bioact. Compat. Polym. 2003, 18, 375−390. (40) Datta, P.; Ghosh, P.; Ghosh, K.; Maity, P.; Samanta, S. K.; Ghosh, S. K.; Mohapatra, P. K. D.; Chatterjee, J.; Dhara, S. In Vitro ALP and Osteocalcin Gene Expression Analysis and In VivoBiocompatibility of N-Methylene Phosphonic Chitosan Nanofibers for Bone Regeneration. J. Biomed. Nanotechnol. 2013, 9, 870−879. (41) Wang, X.; Ma, J.; Wang, Y.; He, B. Bone repair in radii and tibias of rabbits with phosphorylated chitosan reinforced calcium phosphate cements. Biomaterials 2002, 23, 4167−4176. (42) Wang, X.; Ma, J.; Feng, Q. L.; Cui, F. Z. In vivo evaluation of Schitosan enhanced calcium phosphate cements. J. Bioact. Compat. Polym. 2003, 18, 259−271. (43) Guarino, V.; Gloria, A.; Raucci, M. G.; Ambrosio, L. Hydrogelbased platforms for the regeneration of osteochondral tissue and intervertebral disc. Polymers 2012, 4, 1590−1612. (44) Azagarsamy, M. A.; Anseth, K. S. Bioorthogonal click chemistry: an indispensable tool to create multifaceted cell culture scaffolds. ACS Macro Lett. 2013, 2, 5−9. (45) Cha, C.; Liechty, W. B.; Khademhosseini, A.; Peppas, N. A. Designing biomaterials to direct stem cell fate. ACS Nano 2012, 6, 9353−9358. (46) Benoit, D. S.; Schwartz, M. P.; Durney, A. R.; Anseth, K. S. Small functional groups for controlled differentiation of hydrogel-encapsulated human mesenchymal stem cells. Nat. Mater. 2008, 7, 816. (47) Moedritzer, K.; Irani, R. R. The direct synthesis of αaminomethylphosphonic acids. Mannich-type reactions with orthophosphorous acid. J. Org. Chem. 1966, 31, 1603−1607. (48) Kasaai, M. R. Calculation of Mark−Houwink−Sakurada (MHS) equation viscometric constants for chitosan in any solvent−temperature system using experimental reported viscometric constants data. Carbohydr. Polym. 2007, 68, 477−488. (49) Nishimura, S. I.; Tokura, S. Preparation and antithrombogenic activities of heparinoid from 6-O-(carboxymethyl) chitin. Int. J. Biol. Macromol. 1987, 9, 225−232. (50) Blanco, A.; García-Abuín, A.; Gómez-Díaz, D.; Navaza, J. M. Physicochemical characterization of chitosan derivatives. CyTA–J. Food 2013, 11, 190−197. (51) Francis, M. P.; Sachs, P. C.; Elmore, L. W.; Holt, S. E. Isolating adipose-derived mesenchymal stem cells from lipoaspirate blood and saline fraction. Organogenesis 2010, 6, 11−14. (52) Vongchan, P.; Sajomsang, W.; Subyen, D.; Kongtawelert, P. Anticoagulant activity of a sulfated chitosan. Carbohydr. Res. 2002, 337, 1239−1242. (53) Wan, Y.; Creber, K. A.; Peppley, B.; Bui, V. T. Synthesis, characterization and ionic conductive properties of phosphorylated chitosan membranes. Macromol. Chem. Phys. 2003, 204, 850−858. (54) Heras, A.; Rodriguez, N. M.; Ramos, V. M.; Agullo, E. Nmethylene phosphonic chitosan: a novel soluble derivative. Carbohydr. Polym. 2001, 44, 1−8. (55) Hirano, S.; Hasegawa, M.; Kinugawa, J. 13C-NMR analysis of some sulphate derivatives of chitosan. Int. J. Biol. Macromol. 1991, 13, 316−317.

(56) Gamzazade, A.; Sklyar, A.; Nasibov, S.; Sushkov, I.; Shashkov, A.; Knirel, Y. Structural features of sulfated chitosans. Carbohydr. Polym. 1997, 34, 113−116. (57) Lebouc, F.; Dez, I.; Madec, P.-J. NMR study of the phosphonomethylation reaction on chitosan. Polymer 2005, 46, 319− 325. (58) Kuiper, N. J.; Sharma, A. A detailed quantitative outcome measure of glycosaminoglycans in human articular cartilage for cell therapy and tissue engineering strategies. Osteoarthritis and Cartilage 2015, 23, 2233−2241. (59) Orsini, G.; Ruggeri, A.; Mazzoni, A.; Nato, F.; Manzoli, L.; Putignano, A.; Di Lenarda, R.; Tjäderhane, L.; Breschi, L. A review of the nature, role, and function of dentin non-collagenous proteins. Part 1: proteoglycans and glycoproteins. Endodontic Topics 2009, 21, 1−18. (60) Sims, C. D.; Butler, P. E.; Casanova, R.; Lee, B. T.; Randolph, M. A.; Lee, W. P.; Vacanti, C. A.; Yaremchuk, M. J. Injectable cartilage using polyethylene oxide polymer substrates. Plastic and Reconstructive Surgery 1996, 98, 843−850. (61) Tripathi, A.; Kathuria, N.; Kumar, A. Elastic and macroporous agarose−gelatin cryogels with isotropic and anisotropic porosity for tissue engineering. J. Biomed. Mater. Res., Part A 2009, 90, 680−694. (62) Bhat, S.; Tripathi, A.; Kumar, A. Supermacroprous chitosan− agarose−gelatin cryogels: in vitro characterization and in vivo assessment for cartilage tissue engineering. J. R. Soc., Interface 2011, 8, 540−554. (63) Dhiman, H. K.; Ray, A. R.; Panda, A. K. Characterization and evaluation of chitosan matrix for in vitro growth of MCF-7 breast cancer cell lines. Biomaterials 2004, 25, 5147−5154. (64) Lee, K. Y.; Mooney, D. J. Cell-interactive polymers for tissue engineering. Fibers Polym. 2001, 2, 51−57. (65) Ma, P. X. Scaffolds for tissue fabrication. Mater. Today 2004, 7, 30−40. (66) Stenhamre, H.; Nannmark, U.; Lindahl, A.; Gatenholm, P.; Brittberg, M. Influence of pore size on the redifferentiation potential of human articular chondrocytes in poly (urethane urea) scaffolds. J. Tissue Eng. Regener. Med. 2011, 5, 578−588. (67) Oh, S. H.; Kim, T. H.; Im, G. I.; Lee, J. H. Investigation of pore size effect on chondrogenic differentiation of adipose stem cells using a pore size gradient scaffold. Biomacromolecules 2010, 11, 1948−1955. (68) Matsiko, A.; Gleeson, J. P.; O’Brien, F. J. Scaffold mean pore size influences mesenchymal stem cell chondrogenic differentiation and matrix deposition. Tissue Eng., Part A 2015, 21, 486−497. (69) Griffon, D. J.; Sedighi, M. R.; Schaeffer, D. V.; Eurell, J. A.; Johnson, A. L. Chitosan scaffolds: interconnective pore size and cartilage engineering. Acta Biomater. 2006, 2, 313−320. (70) Lien, S.-M.; Ko, L.-Y.; Huang, T.-J. Effect of pore size on ECM secretion and cell growth in gelatin scaffold for articular cartilage tissue engineering. Acta Biomater. 2009, 5, 670−679. (71) Hoemann, C. D.; Sun, J.; Legare, A.; McKee, M. D.; Buschmann, M. D. Tissue engineering of cartilage using an injectable and adhesive chitosan-based cell-delivery vehicle. Osteoarthritis and Cartilage 2005, 13, 318−329. (72) Benya, P. D.; Shaffer, J. D. Dedifferentiated chondrocytes reexpress the differentiated collagen phenotype when cultured in agarose gels. Cell 1982, 30, 215−224. (73) Buschmann, M. D.; Gluzband, Y. A.; Grodzinsky, A. J.; Kimura, J. H.; Hunziker, E. B. Chondrocytes in agarose culture synthesize a mechanically functional extracellular matrix. J. Orthop. Res. 1992, 10, 745−758. (74) Yodmuang, S.; McNamara, S. L.; Nover, A. B.; Mandal, B. B.; Agarwal, M.; Kelly, T. A. N.; Chao, P. H. G.; Hung, C.; Kaplan, D. L.; Vunjak-Novakovic, G. Silk microfiber-reinforced silk hydrogel composites for functional cartilage tissue repair. Acta Biomater. 2015, 11, 27− 36. (75) Nagura, I.; Fujioka, H.; Kokubu, T.; Makino, T.; Sumi, Y.; Kurosaka, M. Repair of osteochondral defects with a new porous synthetic polymer scaffold. J. Bone Jt. Surg., Br. Vol. 2007, 89, 258−264. (76) Gupta, A.; Bhat, S.; Jagdale, P. R.; Chaudhari, B. P.; Lidgren, L.; Gupta, K. C.; Kumar, A. Evaluation of three-dimensional chitosanO


Article

ACS Biomaterials Science & Engineering agarose-gelatin cryogel scaffold for the repair of subchondral cartilage defects: an in vivo study in a rabbit model. Tissue Eng., Part A 2014, 20, 3101−3111. (77) Solchaga, L. A.; Yoo, J. U.; Lundberg, M.; Dennis, J. E.; Huibregtse, B. A.; Goldberg, V. M.; Caplan, A. I. Hyaluronan-based polymers in the treatment of osteochondral defects. J. Orthop. Res. 2000, 18, 773−780. (78) Kapat, K.; Srivas, P. K.; Dhara, S. Coagulant assisted foaming−A method for cellular Ti6Al4V: Influence of microstructure on mechanical properties. Mater. Sci. Eng., A 2017, 689, 63−71. (79) Srivas, P. K.; Kapat, K.; Dhara, S. Process of Dough Forming of Polymer-Metal Blend Suitable for Shape Forming. U.S. Patent US14/ 939,605, November 11, 2015. (80) Kapat, K.; Srivas, P. K.; Rameshbabu, A. P.; Maity, P. P.; Jana, S.; Dutta, J.; Majumdar, P.; Chakrabarti, D.; Dhara, S. Influence of Porosity and Pore-Size Distribution in Ti6Al4V Foam on Physicomechanical Properties, Osteogenesis, and Quantitative Validation of Bone Ingrowth by Micro-Computed Tomography. ACS Appl. Mater. Interfaces 2017, 9, 39235−39248. (81) Kapat, K.; Srivas, P. K.; Dhara, S. Bioactive titanum and/or alloy based nanostructures. Indian Patent 20171123608, July 05, 2017. (82) Kapat, K.; Maity, P. P.; Rameshbabu, A. P.; Srivas, P. K.; Majumdar, P.; Dhara, S. Simultaneous hydrothermal bioactivation with nano-topographic modulation of porous titanium alloys towards enhanced osteogenic and antimicrobial responses. J. Mater. Chem. B 2018, 6, 2877−2893.

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