Augments Growth

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Endogenous Platelet Rich Plasma Supplements/ Augments Growth Factors Delivered via Porous CollagenNanohydroxyapatite Bone Substitute for Enhanced Bone Formation Arun Kumar Teotia, Irfan Qayoom, and Ashok Kumar ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00227 • Publication Date (Web): 30 Apr 2018 Downloaded from http://pubs.acs.org on May 2, 2018

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is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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ACS Biomaterials Science & Engineering

Endogenous Growth

Platelet

Factors

Nanohydroxyapatite

Rich

Plasma

Delivered Bone

Supplements/Augments

via

Substitute

Porous for

Collagen-

Enhanced

Bone

Formation Arun K. Teotia†, Irfan Qayoom† and Ashok Kumar†* †Department of Biological Sciences and Bioengineering; Center for Environmental Science and Engineering; Center for Nanosciences, Indian Institute of Technology Kanpur, Kanpur201806, UP, India

*Corresponding Author. Ashok Kumar, Department of Biological Sciences and Bioengineering, Indian Institute of Technology Kanpur (IITK), Kanpur-208016, UP, India E-mail: [email protected] Phone: +91-512-2594051

1 ACS Paragon Plus Environment

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Abstract Polymer (acrylate) and ceramic bone cements are extensively used as bone void fillers and for implant fixation in orthopedics. These materials have micro- to non-porous architectures. Post implantation they may cause hypoxic and exothermic injuries to already compromised damage site. These materials also have limited interaction with surrounding tissue. In this work we have developed composite collagen-nanohydroxyapatite (CS) bone filler, mimicking porous architecture of trabecular bone. It was functionalized with clinically available bone active agents like bone morphogenetic protein-2 (rhBMP-2) and zoledronic acid (ZA). We investigated synergistic effects of the bone active molecules and endogenous platelet rich plasma (PRP), a source of growth factors on mineralization. Porous CS and collagen/gelatin/chiotosan polymer scaffold (SC) (without nanohydroxyapatite) were synthesized using cryogelation. PRP (10 µL) (~5 × 106 cells), rhBMP-2 (5 µg) and ZA (10 µg) were used to functionalize scaffolds. Bone formation was evaluated at ectopic sites in abdominal pouch and 4.0 mm critical defect in tibia metaphysis of rats. Tissue mineralization was evaluated by micro-CT and histological analysis 12 weeks post-implantation. In vitro cell based studies revealed, PRP functionalization enhances osteoblast proliferation and activity on scaffolds. In vivo BMP+ZA+PRP functionalized scaffolds had higher amount (28 mm3) of mineralized tissue formation as compared to empty defect (20 mm3), suggesting that PRP can augment the osteoinductive properties of functionalized scaffolds both in vitro and in vivo. Enhanced cell infiltration and mineralization can be achieved via CS in comparison to SC, implying their use as porous bone void fillers and substitutes for autografts.

Keywords:

Osteopromotive,

platelet

rich

plasma,

bone-substitutes,

cryogelation,

nanohydroxyapatite, porous scaffold


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Introduction Bone is a highly dynamic tissue capable of regenerating subcritical injuries without any scar tissue formation1, by either filling the defect/void, or establishment of union and bone continuity. Resembling with other tissue, the normal defect healing in bone can also be divided into specific stages such as, clot formation (hemostasis), granulation tissue formation, osteoconduction, wound contraction, followed by new bone formation 1. Fibrin clot formation seals the injured site, by entrapping blood cells and initiating healing process. This is accompanied with release of several leukotrienes and chemoattractants e.g. platelet derived growth factor (PDGF), transforming growth factor-β (TGF-β) and interleukins (ILs), inducing infiltration of leukocytes at the defect site. Additionally, there is infiltration of inflammatory cells such as monocytes, mast cells, polymorphonuclear neutrophils and macrophages. These cells clean the injury site by phagocytosis of bacteria, removing dead tissue and debris. Macrophages also perform clot degradation 2 through secretion of proteases 3

, creating space for new tissue formation. Due to decreased vascularity, lack of blood supply

and degrading clot, the clot site is significantly hypoxic and has low pH than the surrounding tissue. These conditions activate the infiltrated macrophages to secrete angiogenic factors, inducing endothelial cell mediated angiogenesis at the defect site 4. The slowly degrading clot provides 3D surface infiltrated by migrating cells e.g. mesenchymal stem cells, monocytes, fibroblasts, preosteoblasts, chondrocytes, osteogenic cells etc.5,6. They synthesize new matrix leading to defect healing and bone formation through specific pathways (direct/indirect healing)

2,7

osteoblasts

. The infiltrated preosteoblasts gets polarized, differentiating into mature secreting collagen

matrix

(osteoid).

The

secreted osteoid

undergoes

mineralization with bone spicule formation. These spicules grow in size, ultimately fusing together and giving rise to trabecular bone 1. Although autografts are the most preferred choice among surgeons, but due to size limitations and need of secondary surgery, patients 3 ACS Paragon Plus Environment


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often refrain from autograft 8. Most of the currently used bone substitutes have limited osteoinductive cues for cell differentiation and maturation 9. Osteoinductivity limitations with bone grafts and substitutes can be overcome by functionalizing substitutes with bioactive molecules. Bioactive molecules such as; BMPs, TGF, PDGF, VEGF and ILs play important role in different bone defect healing stages

10,11

. TGF-β and PDGFs secreted by entrapped

platelets, are potent mitogens, acting as early phase chemoattractant for inflammatory cells besides stimulating macrophages and osteoblasts

12

. Macrophages and monocytes secrete

fibroblast growth factors (FGFs), IL-1, IL-6 and TNF-α important for chondrocyte proliferation, maturation and remodeling, therefore playing an important role in bone defect healing

10,13

. Differentiated osteoblasts also secrete BMPs (bone morphogenetic proteins)

along with newly deposited matrix 14, inducing further cell differentiation, mineralization and remodeling 15,16. Tayapongsak et al. (1994)

17

for first time and subsequently Marx et al. (1998)

18

have

demonstrated the use of autogenous fibrin glue and platelet rich plasma, respectively in mandibular reconstruction 19. PRP contains high concentration of platelets acting as source of autogenous PDGF, TGF-β1 and TGF-β2. These molecules activates fibroblasts, MSCs, preosteoblasts and endothelial cells, inducing angiogenesis and mineral deposition at the defect site. Additionally, fibrin network formed at the defect also enhances the osteoconduction and bone regeneration 12,17,18. Clot formation along with lack of vascularization leads to disruption of blood supply

20

at

wound site. The nutrients and oxygen transfer via diffusion from capillaries can happen only up to 100-200 µm in tissue matrix

21

leading to oxygen tensions reaching as low as 8.0 mm

Hg 22, leaving wound site largely hypoxic 23. Although, hypoxic conditions at the defect site 22

induces osteogenesis and angiogenesis proliferation and mineralization

, prolonged hypoxia is inhibitory for osteoblasts

20,24,25

. On the other hand, hypoxia strongly stimulates


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osteoclast differentiation 26, enhancing osteoclast activity and bone resorption 26. The hypoxic conditions in the fracture callus were observed until reestablishment of blood supply through neovascularization

13,23,27,28

. Additionally, different cell types in wound communicate via

diffusible signaling molecules secreted in blood for normal tissue dynamics and regeneration. Therefore, for rapid bone formation in filler, a vascular network formation and porous architecture are vital

21,29

. If the nutrient and oxygen supply doesn’t get re-established the

cells may undergo hypoxic injury followed by necrosis

30

. Decrease in BMP2 and Runx2

expression levels were observed in anoxia exposed osteoblasts 24. However, cells pretreated with rhBMP-2 before anoxic assault, had higher Runx2 expression levels, were also able to differentiate and form bone nodules on subsequent culture 24. Blood supply is crucial for bone healing, in situations with disruption of medullary circulation, a temporary devascularization of cortex was observed

31

. Leading to necrosis of bone marrow, unless the blood supply is

restored from periosteum

31

. It was observed that, if the medullary cavity is left open after

reaming, cell infiltration, fibrous network formation followed by osteogenesis and trabecular bone formation occurs, accompanied by bone marrow regeneration medullary cavity was filled with metal pins 33, plaster of paris

34

32

. However, if the

or acrylate cements

34

this

process gets interrupted 31. Calcium phosphate/sulphate based bone cements in their initial phase and acrylate cements throughout their life have very less porosity. Using these cements to fill the large size bone voids or implant fixation, can lead to interruption of blood circulation and anoxia/hypoxia situations at the defect. Hypoxic conditions can persist, until these cements or bone get slowly resorbed, generating pores for neovascularization. Here, in this study we have generated biopolymer-hydroxyapatite based composite supermacroporous scaffolds (CS) and biopolymer alone scaffold (SC) using cryogelation technique. The materials were used alone or loaded with either PRP only, or along with rhBMP-2 and zoledronic acid as bone fillers. It



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has been analyzed whether loading of the substitutes with PRP/rhBMP-2 can maintain higher levels of osteoblast activity and bone formation. The materials were evaluated for enhanced cellular activity in the in vitro studies. Further in vivo studies were carried out either in abdominal pouch (ectopic site) or in critical size sub-metaphysis tibia defect in Wistar rats.

Material and Methods Materials Calcium nitrate and di-ammonium hydrogen phosphate were purchased form S.D. FineChem. (Mumbai, India). Calcium hydroxide, calcium chloride and glutaraldehyde were purchased from Ranbaxy labs (Mumbai, India), rhBMP-2 (Infuse® Bone Graft) was purchased from Medtronic (MN, USA). Zoledronic acid (ZA) (Zometa®, Novartis) was purchased from local pharmacy. Dulbecco’s modified Eagle’s medium (DMEM), 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), β-glycerophosphate, ascorbic acid, SIGMAFASTTM p-Nitrophenyl phosphate (pNpp), trypsin-EDTA, collagen type-I, Alizarin Red-S (alizarin) stain, gelatin and chitosan were all purchased from Sigma Chemical Co. (St. Louis, MO, USA). Fetal bovine serum (FBS) (US origin), phenol red free α-MEM were purchased from Gibco® (MA, USA). Antibiotic cocktail (penicillin and streptomycin) was purchased from Hi-Media (Mumbai, India). Minimum Essential Medium Eagle-alpha modification (α-MEM) was purchased from Thermo scientific (MA, USA). All other chemicals used were of analytical grade. Wistar rats (males) weighing between 250 and 300 g were sourced from Indian Institute of Toxicology Research, Lucknow, India. Synthesis and characterization of nanohydroxyapatite (nHAP) nHAP was synthesized using wet chemical method as described previously

35

using calcium

nitrate tetrahydrate Ca(NO3)2.4H2O (0.96 M) and di-ammonium hydrogen ortho-phosphate (NH4)2HPO4 (0.6 M). The synthesized nHAP was sintered at 800 °C for 4 h to get highly crystalline, phase pure nHAP. Phase purity and particle size analysis was carried out by x-ray 6 ACS Paragon Plus Environment

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diffraction, fourier transform infrared (FTIR) spectroscopy (Spectrum Two, Perkin Elmer, USA) and dynamic light scattering (DLS) (Zetasizer nano ZS 90, Malvern, UK) analysis. Synthesis of macroporous composite scaffolds Macroporous polymer scaffolds were synthesized using cryogelation technology

36

. For

synthesis of polymer scaffold (SC) and polymer-nHAP composite scaffold (CS), chitosan (low viscosity) (1.66% w/v), gelatin (0.6% w/v) and collagen type-I (1% w/v) were dissolved in 1% v/v degassed acetic acid. The polymer blend was either used as such to generate polymer scaffolds (SC), or nHAP (20% w/v) was added to polymer blend for synthesis of composite scaffolds (CS), giving a protein (collagen + gelatin): (nHAP + chitosan) ratio of 1: 13.6 in CS reaction mixture. For cryogelation, glutaraldehyde (0.2% v/v) was used as crosslinker and mixed thoroughly. The polymer blend was poured into tubular molds (2.5 mL syringes) and frozen at -15 °C for 12 h. The cryogels thus formed were thawed at room temperature and washed thoroughly with Milli-Q® water, to remove any unreacted crosslinker. The gels were lyophilized for drying and storage. Characterization of porous scaffolds Physicochemical characterization of the scaffolds was carried out by performing porosity, swelling behavior, flow rate and pore size analysis, as described elsewhere

37,38

. Scanning

electron microscopy (SEM) (EVO-18, Zeiss, Germany) revealed the difference in surface characteristics, pore architecture and pore size of the polymer scaffolds (SC) and nHAP containing composite scaffolds (CS). FTIR (Spectrum Two, Perkin Elmer, USA) analysis was performed to check incorporation of nHAP in the composite scaffolds. nHAP incorporation in scaffold was also carried out by performing TGA analysis (STA 8000, Perkin Elmer, USA) of both CS and SC scaffold and calculating % weight loss in both the scaffolds. Additionally we measured the protein content (gelatin + collagen) in the



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synthesized CS scaffold using bicinchonic acid method as described by Kumar et al.39 using bovine serum albumin as standard. Mechanical characterization of scaffolds Compression analysis of both SC and CS was performed to analyze difference in the mechanical properties of normal polymer scaffold (SC) and that of the composite scaffold (CS). The mechanical analysis was carried out on the cylindrical scaffolds cut in 1: 2 (diameter: height) ratio. The scaffolds were dried by placing in desiccator for 24 h before compression analysis in dry state. The scaffolds were saturated with water by placing in Milli-Q® water for 24 h before analysis. Compressive load was applied to the scaffolds at crosshead speed of 0.6 mm/min, using universal testing machine (UTM), (Instron 3360, Instron, USA). The yield strength was recorded and the compressive modulus was calculated. Blood collection and platelet rich plasma processing Blood was collected from healthy Wistar rats into acid citrate dextrose (ACD) (USP-NF) buffer containing tubes, by retro-orbital puncture under mild anesthesia. ACD was used at 1: 10 (ACD: blood) ratio. After collection, the blood was kept at room temperature and PRP was collected using centrifugation method (Figure S1). Briefly, the blood samples were first centrifuged at 250×g for 15 min at room temperature and the supernatant along with buffy coat was collected and pooled. The pooled supernatant fraction was again centrifuged at 750×g for 15 min and the supernatant was again collected and pooled. The pooled supernatant was again centrifuged at 2000×g for 15 min and the supernatant (platelet poor plasma) was removed. The collected pellet was suspended in the original plasma volume to get platelet rich plasma (PRP) (Figure S1). Normal plasma (10 µl), platelet poor plasma and PRP was placed on three different glass slides, respectively and a smear was obtained. The smear was Giemsa stained to analyze the platelet density in different samples post-


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processing. PRP (500 µl) was obtained from 20 mL of whole blood from healthy rats. The PRP was used immediately in experimental procedures after preparation. Scaffold preparation for three dimensional (3D) cell culture Both SC and CS scaffolds were cut into small circular discs of 2 mm uniform thickness. The scaffold discs were incubated in 70% alcohol for 6 h followed by incubation in 100% alcohol for 2 h, with one alcohol change after one hour to sterilize and dehydrate the scaffolds. The sterile scaffolds were then transferred in tissue culture non-treated 24 well plate, placing one scaffold per each well. The scaffolds were washed twice with 0.01 M PBS and then incubated in PBS along with 2% antibiotic cocktail (penicillin and streptomycin) for 24 h in a humidified incubator at 37 °C with 5% CO2 environment to check sterility. Before seeding the cells, the scaffolds were incubated in complete medium (media supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic cocktail (penicillin and streptomycin)) for 12 h in a humidified incubator at 37 °C with 5% CO2 environment to saturate scaffolds with media. Cell-material interaction studies To check the effect of PRP functionalization of the scaffolds on cell viability and functionality, MC3T3-E1 subclone-4 (mouse pre-osteoblast cells), and Saos-2 (human osteosarcoma), cells were cultured on SC and CS scaffolds either in presence or absence of PRP. For accessing cell viability, MTT assay and for cell functionality alkaline phosphatase (ALP) assay was performed as described elsewhere

35

. For MTT and ALP assay on SC

scaffolds and 2D tissue culture treated plates (TCP), Saos-2 cells were cultured in DMEM complete media, while MC3T3-E1 cells were cultured in α-MEM complete medium supplemented with α-MEM + 10% FBS + β-glycerophosphate (10 mM) + ascorbic acid (50 µg/mL) (differentiation medium). The cells were cultured in a humidified incubator at 37 °C with 5% CO2 environment. For MTT assay on CS scaffolds the cells were cultured using same media as used for SC scaffolds. However, for ALP assay on CS scaffolds the MC3T3-



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E1 cells were cultured on scaffolds in presence of phenol free α-MEM supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic cocktail (penicillin and streptomycin) (complete media) in humidified incubator at 37 °C with 5% CO2 environment, to eliminate interference in ALP assay by phenol red dye. Both Saos-2 and MC3T3-E1 cells were seeded at the seeding density of 1 × 105 cells/well. Cell adhesion and spreading was evaluated by SEM analysis of cells cultured on both SC and CS scaffolds. The SC scaffolds were processed through cryosectioning to analyze osteoinduction under osteogenic conditions, by performing alizarin red S staining for mineralization and bone nodule formation on SC. The cell proliferation and differentiation on CS scaffolds was assessed by performing MTT and ALP assay of MC3T3-E1 cells cultured on CS scaffolds in presence as well as in absence of PRP and differentiation medium. The media was changed after every 48 h. MC3T3-E1 cells cultured on 2D-TCP in presence as well as in absence of PRP and differentiation medium were used as controls. In vivo animal studies In vivo rat abdominal pouch model for osteogenic activity All the animal procedures were performed after approval from the Institute animal ethics committee (IAEC) (reference number IITK/IAEC/2014/1020), following committee for purpose of control and supervision of experiments on animals (CPCSEA) guidelines. A total of 12 male Wistar rats weighing between 300- 350 gm were used. All the animals received two implantations in abdominal muscle (rectus abdominis) pouch, one each on either side of mid line, creating total 24 implantation sites. Animals were divided into four groups having 6 implantation in 3 animals each; the animal groups were as follows: scaffold alone (CS scaffold), (CS + PRP 10 µl/disc), (CS + rhBMP-2 (5 µg/disc) + ZA (10 µg/disc)), (CS + PRP + ZA (10 µg/disc) + rhBMP-2 (5 µg/disc)).


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Square shaped CS scaffold discs were prepared by cutting the scaffolds in 1 cm × 1 cm × 2 mm (length × breadth × thickness) dimensions. Animals were prepared for surgery by intramuscular administration of antibiotic prophylaxis ceftriaxone (40 mg/kg), and removing hairs from abdominal area. For surgery, anesthesia was induced by intraperitoneal administration of ketamine hydrochloride (80 mg/kg) and xylazine hydrochloride (5 mg/kg). The rats were laid in supine position and two 3 cm long incisions were given on either side, 1 cm away from midline exposing the rectus abdominis muscle. A 1.5 cm × 1.5 cm (length × width) pouch was created in abdominal muscle on both sides and the scaffold discs were implanted. The muscle pouch was closed using 5-0 resorbable vicryl sutures, and skin was sutured using 3-0 non-resorbable braided silk sutures. Prophylactic antibiotic therapy was given for 3 days post-surgery. Rats were sacrificed 4 weeks post-implantation and scaffolds were harvested for micro-CT and histological analysis of mineralized tissue. In vivo scaffold implantation in rat tibia defect All animal procedures for rat tibia metaphysis critical defect model were performed after approval from the Institute animal ethics committee (IAEC) (reference number IITK/IAEC/2014/1023), following CPCSEA guidelines. A total of 30 male Wistar rats weighing between 300-350 gm were divided into 10 groups containing 6 animals per group with scaffolds implanted in both the legs (Table 1). Cylindrical discs from lyophilized dry SC and CS scaffolds of 4.5 mm × 5 mm (diameter × height) were prepared by trimming the synthesized cryogel scaffolds to appropriate dimensions. The surgery was performed under anesthesia induced by intraperitoneally administering mixture of ketamine hydrochloride (80 mg/kg) and xylazine hydrochloride (5 mg/kg). All animals were given antibiotic prophylaxis by intramuscular administration of ceftriaxone (40 mg/kg) prior to surgery. The rats were placed in supine position and hairs at the knee area were removed. A 2 cm long incision was given to the skin near the knee area



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starting just below the knee extending towards the tibia shaft. The muscle along with periosteum was removed by scrapping the tibia surface. A 4.5 mm full thickness circular critical size defect was created at tibia metaphysis by slow speed (~1500 rpm) drilling under constant irrigation with sterile saline. The site was flushed with saline to clean debris before scaffold implantation. The scaffolds, pre-saturated with different bioactive molecule combinations according to groups (Table 1), were implanted according to the groups in defect as a filler. In control group, no scaffold was implanted and the defect was left empty. The inner muscle and connective tissue were sutured using 3-0 resorbable sutures and, the skin was sutured with 3-0 nonresorbable sutures to close the wound. Prophylactic antibiotic therapy was continued for next 3 days post-surgery. The animals were free to move around in the cage and had free access to food and water ad libitum. Micro-CT and radiological analysis of defect Ex vivo micro-CT and radiological analysis was performed on the excised tibia samples harvested post-sacrifice. Scanning was performed in Skyscan 1172 (Skyscan 1172, Bruker, Belgium) ex vivo scanner. The scanning was carried out at isotopic voxel size of 10 µm. Misalignment correction, ring artifact correction and image filter (Gaussian, 2 pt. radius) was applied to all the images before reorienting in same plane using data viewer (data viewer, Bruker, Belgium). Image analysis was performed using CTAn (CTAn, Bruker, Belgium) after applying constant thresholding using a circular region of interest (ROI) of 4.5 mm with tapering end; matching to that of drill bit dimensions on all the images to determine highly mineralized tissue volume (BV) deposited at the implant site. Histological analysis for tissue mineralization The tibia samples were fixed in neutral buffered formalin (NBF) for 48 h at 4 °C, and then were transferred to 70% alcohol and incubated at 4 °C until analysed. The fixed samples were divided in two sets, half of the samples were decalcified using Na-EDTA (10%, pH 7.2), the


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decalcified bone was transversely cut into two from center of the defect, paraffin embedded and sectioned at 5 µm thickness for H&E and Masson’s trichrome staining. The remaining undecalcified samples were cut from center of the defect using cutting burr, embedded in methylmethacrylate (MMA) and were sectioned to 6 µm for Alizarin red S staining. Statistical analysis All the in vitro experiments were carried out in triplicate with the minimum sample size of n = 3. For in vivo experiments the sample size was kept at n = 6 for each group. Statistical differences between groups were determined using one way ANOVA and t-test, p value < 0.05. Ethics statement All the animal procedures were performed by following CPCSEA and Institute animal ethics committee (IAEC) guidelines, using the approval number IITK/IAEC/2014/1023 and IITK/IAEC/2014/1020. Utmost care was taken to reduce the animal suffering during the experimentation.

Results Synthesis of calcium deficient nanohydroxyapatite (nHAP) Calcium hydroxyapatite (HAP) synthesized by wet chemical method was white colored, free flowing powder. The HAP synthesized was subjected to thermal treatment by heating at 800 °C for 4 h. When x-ray diffraction pattern of the HAP was compared with ICDD standard for hydroxyapatite (card no. 09-432), almost similar diffraction patterns were observed showing that sintering process lead to phase conversion of HAP, generating phase pure highly crystalline HAP as described previously35. Physicochemical characterization Dynamic light scattering (DLS) based nHAP particle size analysis



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DLS was carried out for particle size and distribution analysis. HAP particles were dispersed in absolute alcohol by sonicating in bath sonicator for 15 min before analysis. The HAP had an average particle size of ~30 nm, with a size distribution ranging from 26 to 32 nm. DLS analysis clearly indicated that HAP is in nano size range with particles having maximum size of 32 nm as described elsewhere previously35. FTIR analysis of nHAP The FTIR vibrational spectra of nHAP pre- and post- thermal treatment at 800 °C for 4 h was performed to check for any degradation and chemical purity of nHAP. Characteristic FTIR spectra was recorded showing a peak at 960 cm-1 (nondegenerate symmetric stretching for PO bond in PO4-), presence of broad peak at 1000-1100 cm-1 characteristic for hydroxyapatite and another broad peak 1041 cm-1 representing asymmetric stretching for PO4- without peaks in other regions validated chemical and phase purity of nHAP (Figure S2). Synthesis of macroporous composite scaffolds Macroporous polymer-nHAP composite scaffolds were synthesized using cryogelation technology 36. Briefly, chitosan (low viscosity) (1.66% w/v), gelatin (0.6% w/v) and collagen type-I (1% w/v) were dissolved in 1% (v/v) degassed acetic acid. The polymer blend was either used as such to generate polymer scaffolds (SC), or 20% (w/v) nHAP was added to synthesize composite scaffolds (CS). In cryogelation the polymer blend is frozen at sub-zero temperatures, this process causes freezing of liquid solvent phase (in present system, water), resulting in cryoconcentration of the dissolved monomer/polymer precursors and crosslinking agent in non-frozen liquid phase (NFLP) (Figure S2). The phenomenon of cryoconcentration of precursors in NFLP leads to gelation/crosslinking and formation of thick pore walls around the embedded ice crystals. Upon thawing the ice crystals melts away leaving behind vacant spaces and pore walls, leading to formation of interconnected macroporous architecture. Physicochemical and mechanical characterization of scaffolds


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CS and SC scaffolds had average porosity of 73.8 ± 5% and 81.5 ± 4% respectively, as estimated by cyclohexane method. On analyzing the swelling behaviour the SC and CS scaffolds had swelling ratio of 25.74 and 5.05, respectively. SEM analysis of the scaffolds revealed incorporation of nHAP into the scaffold matrix and walls, due to which CS had rough surface and much grainy and porous architecture as compared to SC scaffolds which are having smooth walls (Figure 1a-d). It is already known that surface roughness enhances cell adhesion and spreading, the rough surface of the CS might help in enhanced cell adhesion and spreading. Pore size analysis carried out on the SEM images using imageJ

40

revealed that scaffolds are macroporous with average pore size approx. 40-50 µm. The convective flow of solvent defines interconnectivity of the pores, as determined by flow analysis through porous scaffolds, defines ability of porous scaffold to allow blood and nutrient transport. The flow rates were calculated from 1.0 ml/min to 9.0 ml/min. Both CS and SC scaffolds showed excellent flow of water without any back pressure build up upto a flow rate of 6.0 ml/min. This indicates that the gels have interconnected porous architecture providing capability of convective flow with porosity ranging from 70-80% (Table 2). Incorporation of nHAP in polymer scaffold of CS was also analysed by FTIR analysis and TGA analysis. In TGA analysis at 600 °C, the polymer composite (CS) shows only 25.5% weight loss whereas, polymer scaffold (SC) had a weight loss of 70.9%. The significant difference observed in the weight (45%) between two samples, this is due to presence of HAP in CS (Figure S4), which the higher stability of CS at higher temperatures corresponds to thermal resistance of HAP. Protein (collagen + gelatin) incorporated in the synthesized CS scaffold as measured by BCA reagent was 65 µg/mg of the total dry weight of CS scaffold giving a protein (collagen + gelatin): (nHAP + chitosan) ratio of 1: 14.5 in synthesized CS scaffold. A characteristic peak at 960 cm-1 was observed in vibrational spectra of CS which is characteristic for HAP, the same was absent in SC spectra (Figure 1e).



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Compression analysis of the SC and CS was carried out to check the effect of incorporation of nHAP on the mechanical properties of the scaffold. The CS had higher compressive modulus (5.6 MPa in dry state and 0.11 MPa in wet state) than the SC (1.76 MPa in dry state and 0.03 MPa in wet state) confirming that the incorporation of nHAP (20% w/v) had positive effect on the mechanical properties of the scaffolds. Physicochemical properties depict that CS provides rough surface preferred by cells for adhesion and spreading, the presence of nHAP in the scaffold wall matrix provides osteoconductive surfaces. The better mechanical properties allow for ease of handling during implantation in the defect site and it also enables the scaffold to maintain its architecture without collapsing under wet conditions and uniform filling of the defect site without any voids. The porous architecture with interconnected pores of cryogels allows for better blood flow, cell infiltration, vascularization, nutrient and oxygen transport at the implant site, as desired of a large size void filler. Cell material interaction studies To analyze cell adhesion and spreading on SC and CS scaffolds, human osteosarcoma Saos-2 and murine preosteoblast MC3T3-E1 cells were cultured on the scaffolds. Uniform cell spreading on scaffold surface and filopodial extensions of adhesion complex formation were observed in SEM imaging performed 7 days post- cell seeding on the scaffolds (Figure 2). Light microscopy of alizarin red stained Saos-2 cells seeded on 2D TCP and SC seeded with Saos-2 cells, cultured for 8, 14, and 20 days in osteogenic media (osteogenic conditions) shown in Figure 3. Higher amount of mineralization and mineral nodule formation was observed in Saos-2 (osteoblasts) cells at later time points, both on 2D and 3D CS scaffolds, showing positive cell proliferation and mineral deposition. Effect of PRP on cell proliferation was analysed by MTT assay of Saos-2 and MC3T3-E1 cells cultured on the CS in presence of PRP and without PRP, 2D tissue culture plates (TCP) were used as control. On day 1 post-


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seeding TCP as well as CS functionalized with PRP had similar values depicting uniform cell seeding on the scaffolds and TCP. At day 3 initial time point the MTT values were higher on PRP treated CS than CS alone, this might be due to growth factors released by PRP as well as better cell adhesion. Similar results were observed at day 5 time point which might be due to better cell adhesion and proliferation on PRP treated scaffolds. At later time points, day 10, 15 and 20 CS-PRP and CS alone had no significant differences in the MTT values, supporting initial surge in growth might be due to growth factors released from PRP functionalized scaffolds. TCP showed higher values at all the time points as was expected (Figure 4). Effect of PRP on cell differentiation was analysed by culturing MC3T3-E1 cells on CS and CS + PRP scaffolds and analyzing the alkaline phosphatase activity by monitoring ALP values. At initial time point the ALP values were lower in TCP both in presence and absence of osteogenic media. However, the values were higher on CS in presence of osteogenic media (Figure 4b). The cells showed much higher values in presence of osteogenic media at day 10 post- seeding. With cells on 2D TCP in presence of osteogenic media showing ALP values equal to that observed on 3D scaffolds, which were even higher than those observed on 3D scaffolds at later time points. This may be due to higher cell density at later time point as well as the higher stiffness of 2D TCP surface, which also induces cell differentiation into osteoblasts. By day 20 the ALP values were almost similar among all the groups even on 2D TCP in absence of osteogenic media. However, highest ALP values were observed on CS scaffolds functionalized with PRP, in presence of osteogenic media, showing strong osteoinduction. Micro-CT and radiological analysis of the defect In vivo rat abdominal pouch model for osteogenic activity To analyse the osteogenic activity, CS alone or CS functionalized with osteoinductive molecules were implanted in abdominal muscle pouch. On radiographic analysis 4 weeks



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post-surgery, highest amount of mineralized tissue formation was observed in the animals implanted with CS + BMP + ZA followed by CS + BMP + ZA + PRP animals (Figure 5). Similar results were observed on quantitative micro-CT analysis where CS + BMP + ZA showed highest amount of tissue mineralization, followed by CS + BMP + ZA + PRP group. Other animal groups showed very less mineralization, however, group-2 (CS + PRP) animals showed slightly higher mineralization than group-1 (CS alone) animals, but there was no statistically significant difference between group-1 and 2 (Figure 6). Micro-CT studies for tissue mineralization Micro-CT analysis was performed 8 weeks post-implantation, on samples harvested after sacrificing the animals to check for highly mineralized tissue formation and material resorption from the defect site (Figure 7). Micro-CT based histomorphometry was performed to calculate volume of mineralized tissue formed at the defect site. It was observed that bone formation was higher in the animal groups implanted with functionalized composite scaffold (CS). Highest amount of mineralized tissue was formed in CS + ZA + BMP + PRP group (Figure 8), which was higher than empty group and significantly higher (almost double) than the mineralized tissue volume formed in SC + PRP group. In polymer scaffold (SC) groups the defect was largely empty even in functionalized groups with little ossified tissue formation at the periphery of the scaffold. Functionalized SC groups (SC + PRP, SC + BMP + ZA and SC + BMP + ZA + PRP) consistently showed lower amount of mineralized tissue formation in comparison to composite scaffold (CS) groups. The functionalized CS scaffolds shows higher ossification with highest in CS + BMP + ZA + PRP (~ 28 mm3) followed by CS + BMP + ZA groups (27 mm3), which was higher than that observed in empty group (20 mm3) and almost double than that formed in SC + PRP group (16 mm3) (Figure 8). Histological analysis for mineralization


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Hematoxylin & Eosin (H&E) staining H&E staining of decalcified mineralized tissue sections was performed on both CS alone and functionalized CS scaffolds implanted in the abdominal pouch. The mineralized tissue formation was more in both CS + BMP + ZA and CS + BMP + ZA + PRP groups, in comparison to CS alone or CS + PRP group (Figure 9). The functionalized groups also had higher total volume of ossified tissue, with mineralized tissue taking up deep lake color characteristic for mineralized tissue in H&E staining (Figure 9), the mineral nodules were dispersed throughout the mineralized tissue matrix. H&E staining was also performed on the decalcified bone sections from the tibia metaphysis defect implanted with porous scaffold functionalized with PRP along with rhBMP-2 and ZA (Figure 10), 8 weeks post-implantation. There was defect filling observed in empty (control) group where no scaffold was implanted. However, very less trabecular bone formation and thin walled cortical bone formation was observed (Figure 10). In groups implanted with either SC or CS functionalized with bioactive molecules, the bone formation occurred at periphery of defect and at surface of the scaffolds. Higher amount of mineralized tissue formation was observed in groups implanted with functionalized CS. Cell infiltration was observed throughout the scaffold volume (Figure 10). Masson’s trichrome staining Masson’s trichrome staining was carried out on the decalcified tissue sections from abdominal pouch. The bright blue color depicts presence of collagenous soft tissue infiltrated around the implant. Presence of deep blue color (yellow arrow) (Figure 9ii) shows the formation of mineralized tissue. The ossification has occurred in a nodular fashion which are either aggregated or dispersed throughout the tissue matrix (Figure 9ii). In tibia metaphysis defect implanted with the functionalized SC and CS scaffolds, Masson’s staining revealed results similar to that observed in H&E staining. In empty group although



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there was defect bridging observed in the groups, still no complete closure was observed in some animals, also there was very less trabecular bone formation observed at the defect, that too with very thin trabecular walls (Figure 10). In SC implant groups functionalized with PRP, BMP + ZA or PRP along with BMP + ZA, lowest amount of mineralized tissue formation was observed in SC + PRP group followed by SC + BMP + ZA group. The SC + BMP + ZA + PRP group showed highest amount of mineralized tissue formation, with new bone formation observed in blue color at the defect site (Figure 10). The animals implanted with functionalized composite scaffolds (CS) showed higher amount of mineralized tissue formation when compared to empty groups or with functionalized SC implanted groups. The CS functionalized with BMP and ZA either alone or with PRP had higher amount of mineralized tissue formation than SC functionalized in similar combinations. This reveals that osteoconductive surface of CS leads to better cell infiltration, differentiation and mineralization. When animals were given 10 µg ZA systemically (one-time dose) along with CS alone (CS + ZA(I)) or CS + PRP (CS + PRP + ZA(I)) implanted, although there was less mineralization in these groups compared to other groups, still it was higher than that observed in SC + PRP and empty group. These results further supports that CS provides osteoconductive surface for better cell infiltration and mineralization. Among functionalized CS groups, BMP functionalized scaffolds always had higher bone formation with highest degree of mineralization observed in CS + BMP + ZA + PRP group followed by CS + BMP + ZA and CS + BMP + PRP groups. Thin bridging was observed in CS + BMP + PRP group, which might be due to better cell adhesion and migration on PRP treated scaffolds. The results, illustrate that CS can act as carrier for BMP and ZA and slowly release it at the defect site leading to higher concentration of osteopromotive molecules at the defect site for sustained period. This was also observed by consistent presence of higher amount of recently


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mineralized tissue (blue color) deposited at defect site in CS functionalized with BMP and BMP along with ZA. Almost comparable amount of mineralization was observed in CS + BMP, CS + BMP + ZA and CS + BMP + PRP groups (Figure 10) but was lesser in CS + PRP + ZA(I) groups. This illustrates that PRP under osteogenic conditions in presence of osteoinductive factor (BMP) enhances bone formation, but in non-osteogenic environments does not induce ossification by itself similar to ZA. Alizarin Red S staining When alizarin red staining was performed on the undecalcified sections of tissue harvested 4 weeks post-implantation into the rat abdominal pouch model (Figure 11), the CS alone and CS + PRP groups had almost negligible ossified nodule formation post-implantation and the scaffold was in elongated flat shape. However, in CS + BMP + ZA and in CS + BMP + ZA + PRP groups the scaffold gets integrated with newly developing tissue and new round shaped mineralized tissue got deposited at the defect site, which was quite different than that implanted initially and that in the CS alone and CS + PRP groups. Highest amount of mineralized tissue formation in nodular focal sites was observed in CS + BMP + ZA + PRP group followed by CS + BMP + ZA group (Figure 11). The results were matching to that observed in the micro-CT studies. In the alizarin red staining of rat tibia bone sections implanted with porous scaffolds and harvested 8 weeks post-implantation, highest amount of mineralized tissue formation was observed in CS + BMP + PRP + ZA group followed by CS + BMP + ZA group. None of the groups showed complete defect healing and bridging at the defect site. In empty groups though there was bridging observed, but the defect was largely empty with very less amount of thin walled trabecular bone formation observed. Functionalized CS implant groups had higher amount of bone formation compared to the SC groups where the defect was largely empty with very less alizarin positive nodular bone formation compared to CS groups



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(Figure 12) which is similar to Masson’s staining results. The lateral sections of undecalcified functionalized SC groups (SC + BMP + ZA, SC + BMP + ZA + PRP) and CS groups (CS + BMP + ZA, CS + BMP + ZA + PRP) stained with calcium positive alizarin (Figure 13) also illustrated similar results, the tissue mineralization initiated from the periphery of the defect inwards in both the groups. In CS scaffold groups higher amount of mineralization was observed compared to SC groups, CS + BMP + ZA + PRP groups had higher mineralization than SC + BMP + ZA + PRP group, this might be due to better cell infiltration and mineralization in CS groups due to presence of osteoconductive surface.

Discussion Similar to other tissues, bone injury healing also initiates with fibrin clot formation and sealing of the defect inhibiting blood loss

1,2

. This if followed by secretion of

chemoattractants from entrapped inflammatory cells, enabling further cell migration, defect site cleaning and repair 2,4. Clot formation and sealing of the defect site is beneficial at initial stages. However, clot formation also inhibits cell migration into the wound and inhibits exchange of nutrient and gases. Inhibition of nutrient availability, to metabolically demanding regenerating tissue along with hypoxic stress, often leads to tissue necrosis 2,4. In bone this necrotic tissue undergoes apoptosis and gets replaced as part of healing process 41,42

, by newly deposited tissue. The tissue undergoes mineralization along with angiogenesis

restoring blood supply to the tissue. However, in critical size injuries where surgical intervention is required and bone substitutes and bone cements are used as fillers, this process gets slowed down, in case of slowly self-resorbing fillers and cements. If the filler is metal or polymer cement which are non-resorbing and non-porous materials, the normal healing process doesn’t take place resulting in necrosis of bone and inhibition of defect filling 31. As part of healing process, tissue necrosis and bone loss persists until establishment of blood 22 ACS Paragon Plus Environment

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circulation through neo-vasularization. Although calcium sulphate-calcium phosphate (hydroxyapatite) based biphasic cements are self-resorbing, generating porous matrix at implant site by slowly leaching away at later time points. At the time of initial setting, these materials are microporous and may not allow blood circulation at the defect site

35,43

. In case

of autografts, the vascularized bone grafts provides both porous vascularized osteoconductive surface as well as several growth factors, which is key reason for the success of autografts and vascularized grafts 44,45. In the present study, we have synthesized composite porous scaffolds using a combination of biopolymers (collagen, gelatin and chitosan) to generate polymer scaffold (SC). Nanohydroxyapatite was added to the polymer solution to generate porous composite scaffolds (CS), using cryogelation technology. The porous scaffolds were used either alone or after functionalization with bioactive factors in animal models to analyze bone formation. The synthesized nHAP was sintered at 800 °C to enhance its crystallinity and phase purity, which were confirmed by FTIR and XRD analysis. The synthesized nHAP was used along with biopolymers to generate composite scaffolds. The synthesized scaffolds were macroporous in nature and architecture similar to trabecular bone, with porosity ranging from 70-80%, with SC and CS having swelling ratio of 25.74 and 5.05, respectively showing high swelling behaviour, appropriate for completely filling the defect site post implantation. On mechanical characterization SC had a compressive modulus of 1.76 MPa and that of CS was 5.6 MPa. Under wet conditions both scaffolds showed decrease in mechanical stiffness with compressive modulus of 0.03 MPa and 0.11 MPa for SC and CS, respectively. This might be due for higher amount of nHAP incorporated into the CS scaffolds providing higher compressive strength as well as thermal stability, as observed through TGA analysis. Under both wet and dry conditions CS has better mechanical properties which will provide ease in handling and workability, and prevent scaffold from collapsing. Both scaffolds were



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biocompatible in nature supporting Saos-2 and MC3T3-E1 cell proliferation on surface, as observed through MTT and SEM analysis, with cells growing with extended morphology. Cell proliferation was also analysed by MTT assay of MC3T3-E1 and Saos-2 cells cultured on PRP functionalized (SC + PRP and CS + PRP) scaffolds. The PRP functionalization enhanced cell growth during initial period up to day-5 on treated scaffolds, this might be due to released growth factors and better cell adhesion. ALP assay of MC3T3-E1 cells cultured on CS scaffold in presence or absence of PRP under either osteogenic or non-osteogenic conditions, revealed highest amount of ALP expression by cells seeded on CS functionalized with PRP under osteogenic media. From our previous work on nanocement chitosan + agarose + HAP composite porous scaffold

35

and silk +

14

, we concluded that composite

scaffold can act as carrier for bone active molecules (BMP) and induce bone formation 14,46. This early delivery of BMP at the defect site can maintain higher osteogenic activity and differentiation of progenitor cells into osteoblasts leading to higher levels of mineralized tissue formation. We synthesized porous polymer composite scaffold using collagen-I and gelatin without HAP (SC) and along with nHAP (CS). As collagen is natural component of bone and higher concentrations of nHAP will provide more stiffness as well as stronger osteoconductive cues to the scaffold 6. Gelatin was incorporated as it is degradation product of collagen and provides RGD motifs for cell adhesion, additionally gelatin also enhances degree of crosslinking and mechanical strength of the scaffolds 47. Chitosan was added as it enhances the viscosity of polymer solution helping in maintaining homogeneity of dispersed hydroxyapatite along with providing mechanical stiffness. Additionally, chitosan is known for its antimicrobial properties. To analyse the carrier as well as osteoconductive properties, CS either alone or functionalized with PRP, BMP and ZA were implanted into rectus abdominis muscle pouch in Wistar rat to check for ectopic bone formation. This study also


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led us to evaluate the effect of autogenous PRP as a source of osteoinductive factors. MicroCT analysis performed 4 weeks post-implantation revealed that there is no significant difference in total amount of mineralized tissue formed among CS + BMP + ZA and CS + BMP + ZA + PRP groups However, it was higher than that formed in CS + PRP alone group. Also the CS + PRP group had no significantly higher amount of bone formed in comparison to CS alone at ectopic site. This reveals that PRP alone does not have a strong osteoinductive activity, compared to that observed with BMP-2. However, due to its vascularization activity and weak osteogenic activity, it can enhance overall mineralization without any negative effects. On histological analysis, also CS + BMP + ZA and CS + BMP + ZA + PRP both implants had higher net mineralized tissue volume. The CS + BMP + ZA + PRP group had bigger size mineral nodules deposited in comparison to CS + BMP + ZA which had small sized numerous dispersed mineral nodules throughout the implant, leading to almost equal volumes of total mineralized tissue formation. A therapy giving results similar to CS + BMP + ZA + PRP with bigger size mineral nodule formation will be preferred over smaller sized numerous nodules as with CS + BMP + ZA, which may resorb readily. To check the efficacy of composite porous scaffold as carrier for bioactive molecules and bone void filler, SC and CS scaffolds were implanted in the critical size tibia defect either alone or after functionalization with PRP alone or along with BMP and ZA. Micro-CT analysis carried out 8 weeks post-implantation on excised bone revealed, that functionalized CS consistently formed higher volume of mineralized tissue in comparison to non-functionalized CS. This was always higher than that formed with non-functionalized as well as functionalized polymer scaffold (SC). With CS + BMP + ZA + PRP and CS + BMP + ZA group having almost double the amount of mineralized tissue formed at the defect sites in comparison to SC + PRP group. This was also higher than the volume formed in SC + BMP + ZA and SC + BMP + ZA + PRP groups. Empty group had mineralization higher than that observed in the



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SC + PRP group, and almost equal to the volume observed in SC + BMP + PRP and SC + BMP + ZA + PRP groups. This depicts that SC is not able to release bioactive molecules in controlled manner, and is no better than an empty defect. However, both micro-CT as well as histology results depicted only thin walled cortical bridging in the empty group, with very less trabecular bone formation and defect filling, leaving defect largely empty. The histology results in both decalcified H&E and Masson’s stained as well as undecalcified alizarin stained bone sections also portrayed similar results. All the BMP functionalized CS scaffolds showed higher amount of new mineralized tissue (deep blue color) deposited in and around the defect. This pattern was not observed in non BMP functionalized groups and in SC, showing that CS is acting as better carrier for BMP and releasing it slowly in sustained manner. This constant induction leads to higher amount of mineralized tissue formation at the defect site. The SC + PRP, CS + ZA and CS + PRP + ZA had lower amount of new mineralized tissue formation in comparison to BMP functionalized SC and CS groups. This depicts that PRP alone without BMP does not have strong osteoinductive activity. Similar results were observed in alizarin stained undecalcified bone sections, where higher amount of mineralization was observed in CS + BMP + ZA + PRP group followed by CS + BMP + ZA group. The rest of the groups showed even lesser amount of mineralization and defect filling leaving defect largely empty even after 8 weeks post-implantation. The ZA functionalized groups also showed higher mineral density deposited at defect site in comparison to SC + PRP, CS + BMP and CS + BMP + PRP groups, which may be due to ZA mediated inhibition of osteoclast mediated bone resorption and controlled release of ZA from CS. The lateral sections of undecalcified bone samples also shows similar trend with new mineralized tissue being deposited from periphery inwards. Higher mineral volume formation was observed in CS + BMP + ZA and CS + BMP + ZA + PRP groups in comparison to SC + BMP + ZA and SC + BMP + ZA + PRP groups. This shows CS scaffold acts as better carrier for ZA and


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BMP due to higher amount of HAP (calcium) present in CS in comparison to SC, which may be due to inherent affinity of both BMP and ZA towards HAP. Thus, functionalized composite scaffolds can better act as filler for large size bone defects than the non-composite and non-functionalized scaffolds, also PRP functionalization due to its angiogenic activity can induce early vascularization and enhanced mineralized tissue deposition. This early vascularization can protect the already compromised defect site from anoxic necrotic injury, also enabling enhanced mineralized tissue formation.

Conclusion In this study, we have demonstrated that the porous composite scaffold (CS) provides osteoconductive surfaces for osteogenic cell infiltration and mineralized matrix deposition. The porous composite scaffolds loaded with bone active molecules such as BMP and ZA acts as better carrier for BMP and ZA in comparison to the polymer scaffolds (SC) without HAP. Composite scaffold provide porous osteoconductive surface for early cell infiltration and neovascularization, leading to enhanced bone formation. Autogenous PRP acts as an easily available source of bioactive growth factors which can augment the osteoconductive properties of the scaffolds by enabling better cell adhesion and supporting neovascularization. The functionalized porous scaffolds can be used as fillers in large size bone defects in nonload bearing situations, without compromising defect site connectivity, oxygen and blood circulation thereby enhancing tissue formation, mineralization and rapid defect healing. These porous composite scaffolds along with nanocement can be used as filler for large size critical bone defects, overcoming autograft size limitations. Depicting that bioactive molecule functionalized composite scaffolds can efficiently and effectively substitute autografts as fillers, especially in non-load bearing bone defects.

Supporting Information 27 ACS Paragon Plus Environment


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Information related to FTIR based purity analysis of synthesized nano-hydroxyapatite. Porous scaffold synthesis using cryogelation, TGA based HAP incorporation and residual mass analysis and process for generation of platelet rich plasma (PRP) from whole blood using simple centrifugation method.

Acknowledgements The authors acknowledge Department of Biotechnology (DBT) (DBT-VINNOVA, IndoSwedish project BT/IN/Sweden/08/AK/2017-18) and Department of Science and Technology (DST), (DST-VR, Indo-Swedish project), Ministry of Science and Technology, Govt. of India for financial support. AKT, and IQ acknowledges Ministry of Human Resources and Development, Govt. of India and IIT Kanpur for PhD fellowships and AK acknowledges DBT TATA Innovation Fellowship, Govt. of India.

Author contributions AK and AKT designed the whole study and developed and characterized the materials. AKT performed the animal surgery and histology. AKT and AK performed the ex vivo micro-CT and image analysis. AK, AKT, IQ planned the in vivo animal experiments and performed result interpretation. AKT wrote the MS draft with inputs from IQ and AK. Authors would also like to acknowledge Mr. Deepak Bushan Raina, Lund University, Sweden for discussion and inputs in the study.

Disclosure This work is accepted for presentation at 59th biannual Nordic orthopedic foundation (NOF) congress-2018, to be held in Reykjavik, Iceland. This work is filed for Indian provisional invention disclosure with patent application number 201811015012; dated 20-04-2018.


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Conflict of interest All other authors declare no conflict of interest related to this study.

Abbreviations BMP, bone morphogenetic protein; ALP, alkaline phosphatase; PRP, platelet rich plasma; micro-CT, micro-computer tomography; HAP, hydroxyapatite; CS, calcium sulphate; CP, calcium phosphate; TCP, tri-calcium phosphate; DMEM, Dulbecco’s modified Eagle’s media; EDTA, ethylenediamine tetraacetic acid; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium

bromide);

βGP,

β-Glycerophosphate;

DAPI,

(4',6-diamidino-2-

phenylindole); H&E, Hematoxylin and Eosin; COL-I, collagen type-I; MMA, methyl methacrylate; SEM, scanning electron microscopy; TEM, Transmission electron microscopy; NMR, Nuclear magnetic resonance; EDS, energy dispersive x-ray spectroscopy; BSE, Backscattered electron; XRD, X-ray diffraction spectroscopy; BV, bone volume; TV, total volume; ICDD, international center for diffraction data; TCP, tissue culture plate; FTIR, Fourier transform infrared spectroscopy.



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List of Tables:

Table 1: Different animal groups and scaffolds implanted in tibia defect Group No.

Implantation in left leg

Implantation in right leg

Group-1 & 2

SC+PRP

Empty

Group-3 & 4

SC+BMP+ZA+PRP

SC+BMP+ZA

Group-5 & 6

CS+PRP+ZA(I)

CS+ZA(I)

Group-7 & 8

CS+BMP+PRP

CS+BMP

Group-9 & 10

CS+BMP+ZA+PRP

CS+BMP+ZA



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Table 2: Different physicochemical parameters of SC and CS porous scaffolds Density (gm/cm3)

Swelling ratio

Porosity (%)

Polymer Scaffold (SC)

0.046

25.74

81.6

Composite Scaffold (CS)

0.209

5.05

73.8

Scaffolds


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List of Figures:

Figure 1: Physicochemical characterization of porous scaffolds. Scanning electron microscope (SEM) images of (a, b) polymer alone scaffold (SC) and (c, d) polymer + nHAP (composite) scaffold (CS) formed by cryogelation; (b) SC is having smooth walls whereas; (d) CG is having rough and porous walls; (e) FTIR spectra of different scaffolds, presence of characteristic peak at 960 cm-1 in CS shows incorporation of HAP in scaffold. (Magnification 5,000×)



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Figure 2: Scanning electron microscope (SEM) analysis of cells growing on porous CS scaffold. (a) SEM image of MC3T3-E1 preosteoblast cells cultured on composite scaffold (CS) after 7 day culture; (b) false colored image showing cells growing over porous scaffold surface forming extensive filopodia extensions (inset yellow box).


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Figure 3: Saos-2 cells seeded on two dimensional (2D) plates and on three dimensional (3D) CS scaffolds. (a) Saos-2 cells seeded on 2D TCP stained with alizarin red (red) and counter stained with fast green (green) to check mineral nodule deposition; (b) Saos-2 cells seeded on 3D CS scaffolds stained with alizarin red (red) to check mineral nodule formation.



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Figure 4: Cell-material interaction studies on CS scaffolds. (a) Graph representing the cell proliferation analysis by MTT assay of cultured MC3T3-E1 and Saos-2 cells on CS in presence and absence of PRP; (b) graph representing ALP values of MC3T3-E1 cultured on CS scaffolds in presence of PRP and 2D TCP were used as control (mean ± SD).


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Figure 5: Radiological and micro-CT analysis of mineralized tissue formation. Radiological and micro-CT images of mineralized tissue formed among different groups in abdominal pouch model (ectopic site) (a-d).



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Figure 6: Micro-CT based quantitative histomorphometric analysis of mineralized tissue formation. Graph representing micro-CT based quantitative histomorphometric analysis of ectopic mineralized tissue formation in abdominal pouch model among different groups (n = 6, mean ± SD).


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Figure 7: Micro-CT radiographic analysis of tibia defect in animal model. Micro-CT images of the defect in 8 weeks post-implantation showing mineralized tissue formation among different groups; and micro-CT 3D rendered images of the corresponding groups showing 3D model of the defect site showing morphology and mineralized tissue formed at the defect site.



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Figure 8: Mico-CT based mineralized tissue formation analysis. Micro-CT radiographic analysis of the defect site and graph representing micro-CT quantitative histomorphometric analysis of mineralized tissue formation in different groups (p value < 0.05).


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Figure 9: H&E and Masson’s trichrome staining for mineralized tissue formation. (i) H&E staining of the implanted functionalized scaffold from different groups, showing highly mineralized tissue formation (yellow arrow); (ii) Masson’s trichrome staining for highly mineralized tissue formation (yellow arrow).



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Figure 10: H&E and Masson’s trichrome staining of tibia implanted with porous scaffold. In Masson’s trichrome image dark magenta color is representing old bone, whereas the blue color (collagen) is representing freshly mineralized osteoid, which is higher in BMP functionalized groups.


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Figure 11: Alizarin red staining of CS scaffold 4 weeks post-implantation. Alizarin red staining of undecalcified tissue sections of the implanted scaffold 4 weeks postimplantation showing formation of calcified tissue (red) counter stained fast green color represents surrounding soft tissue.



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Figure 12: Representative images of alizarin red stained sections of undecalcified rat tibia. Alizarin red staining of rat tibia implanted with porous scaffolds, 8 weeks postimplantation, showing mineralized tissue formation among different groups.


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Figure 13: Alizarin red stained lateral sections of undecalcified rat tibia implanted with porous scaffolds. Representative image of undecalcified lateral sections of rat tibia implanted with functionalized porous polymer scaffold (SC) and composite scaffold (CS) 8 weeks post-implantation, showing implant site.



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For Table of Content Use Only

Endogenous Platelet Rich Plasma Supplements/Augments Growth Factors Delivered via Porous Collagen-Nanohydroxyapatite Bone Substitute for Enhanced Bone Formation Arun K. Teotia†, Irfan Qayoom† and Ashok Kumar†* †Department of Biological Sciences and Bioengineering; Center for Environmental Science and Engineering; Center for Nanosciences, Indian Institute of Technology Kanpur, Kanpur201806, UP, India

Graphical Abstract: Schematic representation of implantation of functionalized porous composite scaffold as bone filler in tibia bone defect model


Augments Growth

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