Cross-correlative 3D micro-structural investigation of human bone processed into bone allografts

Materials Science and Engineering C 62 (2016) 574–584

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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec

Cross-correlative 3D micro-structural investigation of human bone processed into bone allografts Atul Kumar Singh a, Astrid Lobo Gajiwala b, Ratan Kumar Rai c, Mohd. Parvez Khan d, Chandan Singh c, Tarun Barbhuyan d, S. Vijayalakshmi a, Naibedya Chattopadhyay d, Neeraj Sinha c,⁎, Ashutosh Kumar e,⁎⁎, Jayesh R. Bellare a,f,⁎⁎⁎ a

Centre for Research in Nanotechnology & Science, Indian Institute of Technology Bombay, Mumbai 400076, India Tissue Bank, Tata Memorial Hospital, Parel, Mumbai 400012, India c Centre of Biomedical Research, SGPGIMS Campus, Lucknow 226014, India d Division of Endocrinology, Center for Research in Anabolic Skeletal Targets in Health and Illness (ASTHI) CSIR-Central Drug Research Institute, Lucknow 226031, India e Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Mumbai 400076, India f Department of Chemical Engineering, Indian Institute of Technology Bombay, Mumbai 400076, India b

a r t i c l e

i n f o

Article history: Received 18 November 2015 Received in revised form 22 January 2016 Accepted 2 February 2016 Available online 3 February 2016 Keywords: Human bone Allograft Solid state NMR Scanning electron microscopy Bone Processing MIMICS

a b s t r a c t Bone allografts (BA) are a cost-effective and sustainable alternative in orthopedic practice as they provide a permanent solution for preserving skeletal architecture and function. Such BA however, must be processed to be disease free and immunologically safe as well as biologically and clinically useful. Here, we have demonstrated a processing protocol for bone allografts and investigated the micro-structural properties of bone collected from osteoporotic and normal human donor samples. In order to characterize BA at different microscopic levels, a combination of techniques such as Solid State Nuclear Magnetic Resonance (ssNMR), Scanning Electron Microscope (SEM), micro-computed tomography (μCT) and Thermal Gravimetric Analysis (TGA) were used for delineating the ultra-structural property of bone. ssNMR revealed the extent of water, collagen fine structure and crystalline order in the bone. These were greatly perturbed in the bone taken from osteoporotic bone donor. Among the processing methods analyzed, pasteurization at 60 °C and radiation treatment appeared to substantially alter the bone integrity. SEM study showed a reduction in Ca/P ratio and non-uniform distribution of elements in osteoporotic bones. μ-CT and MIMICS® (Materialize Interactive Medical Image Control System) demonstrated that pasteurization and radiation treatment affects the BA morphology and cause a shift in the HU unit. However, the combination of all these processes restored all-important parameters that are critical for BA integrity and sustainability. Cross-correlation between the various probes we used quantitatively demonstrated differences in morphological and micro-structural properties between BA taken from normal and osteoporotic human donor. Such details could also be instrumental in designing an appropriate bone scaffold. For the best restoration of bone microstructure and to be used as a biomaterial allograft, a step-wise processing method is recommended that preserves all critical parameters of bone, showing a significant advancements over currently existing methods. © 2016 Elsevier B.V. All rights reserved.

1. Introduction The use of bone allografts (BA) in the reconstruction of skeletal defects is a well-established medical practice. While bone obtained from deceased donors form the bulk of banked tissues, femoral heads obtained as surgical residues from osteoporotic patients undergoing hip joint

⁎ Corresponding author. ⁎⁎ Corresponding author. ⁎⁎⁎ Correspondence to: J. R. Bellare, Department of Chemical Engineering, Indian Institute of Technology Bombay, Mumbai 400076, India. E-mail addresses: [email protected] (N. Sinha), [email protected] (A. Kumar), [email protected] (J.R. Bellare).

http://dx.doi.org/10.1016/j.msec.2016.02.002 0928-4931/© 2016 Elsevier B.V. All rights reserved.

replacement surgeries have also been found to be clinically useful, particularly when morselized (granules/chips) and used in revision hip or maxillofacial surgeries [1,2]. All such BA however, carry the risk of disease transmission from donor to recipient and has the potential to cause immune reactions in the recipient [3,4]. To reduce these risks, Tissue Banks complement stringent donor screening with good manufacturing practice, compliance with standards developed by professional organizations (such as AATB, APASTB) for controlled environments and various protocols to eliminate bacterial and viral contamination [5]. The Tata Memorial Hospital (TMH, Mumbai) Tissue Bank uses a combination of jet lavage, pasteurization at 60 °C, ethanol soaks and terminal sterilization with 25 kGy of gamma radiation to ensure the safety

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of its BA [6]. The BA are either freeze-dried and stored at room temperature or frozen at −80 °C. While the effects of all these processes on the biomechanical and biological properties of BA [7,8] and on their immunogenicity and subsequent incorporation [9] have been widely studied, however studies on the micro-structural changes are limited. The unique property of bone is due to the state of aggregation and relative arrangement of the various constituents. This is a multi-scale phenomenon, requiring analyses at molecular, nanometer and micrometer scales. Thus knowing about the changes occurring in the different processing steps of allograft processing would establish the fundamental basis and also provide insight into further improvements in making the bone allograft of bone scaffolds for clinical uses. That is why a wide combination of techniques was used for delineating ultrastructural property of bone. The aim of this study was to analyze three-dimensional microstructural changes in BA collected from normal and osteoporotic bone donor processed into BA using the protocols developed in-house by the TMH Tissue Bank. Changes in the bone microstructure at each stage of processing and in the final BA used for transplantation were examined in order to elucidate the effects of processing on banked bone. In order to be comprehensive, a wide combination of techniques was used. This includes Solid State Nuclear Magnetic Resonance (ssNMR), Scanning Electron Microscope (SEM), Thermal Gravimetric Analysis (TGA) and micro-computed tomography (μCT), because they are the best for delineating ultra-structural property of bone. Proton 1D and 1 H-13C cross-polarization (CP) NMR was used to measure relative water content and the structural integrity of the collagen respectively. 31 P T1 (longitudinal relaxation) was used to monitor the local crystallinity of inorganic part of the BA, whereas, 1H–31P heteronuclear correlation experiment was used to probe local water environment near 31P (within 1 nm). The combination of these experiments covers a wide range of the structural aspect of BA as it reports changes in water content, and organic and inorganic component of BA. To get coherence between the techniques used, a correlation analysis was performed between the NMR parameters representing BA biomaterial integrity at the atomic scale, and an established set of parameters, including micro-architecture by SEM and MIMICS®. The aim was to investigate whether the NMR parameters could serve as the surrogate of any of the later. Taken together, our study helps to elucidate the effects of processing on banked BA. Additionally it describes the ultra-structure of BA thereby providing a basis for developing and evaluating bone scaffolds. 2. Experimental section 2.1. Background to bone processing One of the basic steps in bone processing used in many tissue banks is the removal of bone marrow and cell debris with washing under pressure or the use of various fluids or detergents. This not only improves the osteoconductive capacity of bone [10], but also reduces the antigenicity, bacterial and viral contamination. A simple means of closing the window of possible viral infections is pasteurization at 60 °C. Incubation for 30 min in a water bath at 56 °C inactivates HIV [11], and HIV reverse transcriptase activity reaches zero after 20 min of incubation at 56 °C [11]. Such treatment does not affect the osteogenic properties of the bone graft [12]. Ethanol soaks are also used to reduce the potential viral repository as they have been shown to inactivate the coated viruses such as HIV and the hepatitis viruses [13]. In addition alcohol serves to remove lipids, which inhibit graft incorporation [14] particularly when radiation is used for terminal sterilization, as irradiation can cause medullary lipids to release compounds that are toxic for osteoblast-like cells [15]. Gamma radiation of bone grafts is a preferred means of terminal sterilization because of its ability to penetrate compact bone uniformly. The dose used to eliminate bacteria and viruses varies from 15 kGy to

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25 kGy although higher doses may be used to eliminate viruses [16]. Doses higher than 25 kGy have been shown to compromise the biomechanical properties of allografts [17] increasing the risk of fracture and affecting the load bearing capacity in structural grafts [8]. Among the methods used for the preservation of bone for prolonged periods of time are deep freezing and freeze-drying. Both these methods also reduce the antigenicity of bone [18]. Freeze-drying has the additional advantage of permitting convenient storage at room temperature. However, it has a detrimental effect on the mechanical strength of the graft, but this can be reasonably restored with rehydration prior to transplantation. For our study, normal human bone samples were obtained from an amputated tibia of a patient with a soft tissue sarcoma where MRI scans had confirmed that the bone was uninvolved. Osteoporotic femoral heads were obtained as surgical residues from clinically diagnosed osteoporotic patients undergoing hip joint replacement surgery. These bones were procured from the TMH Tissue Bank, Mumbai, India. Approval for the project (No. 1007) was obtained from the Institutional Review Board of TMH vide letter dated 29th March, 2012. Patient consent was obtained in each case for the use of the bone for research purposes. The samples for the study were treated as follows: Bone samples, 3 mm × 10 mm were cut from the tibia [normal cortical (Sample code: NCo) and cancellous bone (Sample code: NCa)] and the femoral heads (osteoporotic cortical (Sample code: OCo) and cancellous bone (Sample code: OCa)). Samples were washed to remove blood, bone marrow and soft tissue using jet lavage. Each sample group contained normal cortical and cancellous bone, and osteoporotic cortical and cancellous bone. The groups were processed as follows: Process 1 (code: U for Unprocessed): The cleaned bones were packaged in polyethylene sleeves in a laminar airflow cabinet. The samples were stored at −80 °C. Process 2 (code: P for Pasteurized): The cleaned bones were pasteurized at 60 °C for 3 h followed by vigorously washing and packaging in polyethylene sleeves in a laminar airflow cabinet. The samples were stored at −80 °C. Process 3 (code: A for Alcohol): The cleaned bones were placed in 70% ethanol for 1 h. They were then thoroughly washed and packaged in polyethylene sleeves in a laminar airflow cabinet. The samples were stored at −80 °C. Process 4 (code: FD for freeze-dried): The cleaned bones were freeze-dried and stored at room temperature. Process 5 (code: R for Radiation): The cleaned bones were double packed in polyethylene containers in a laminar airflow cabinet and irradiated using 25 kGy of gamma radiation. The samples were stored at −80 °C. Process 6 (code: F for Full Processing): The cleaned bones were pasteurized at 60 °C for 3 h. They were then vigorously washed and placed in 70% ethanol for 1 h. After thorough washing they were stored at − 80 °C and finally freeze-dried to remove 95% of the moisture. The bones were terminally sterilized with 25 kGy of gamma radiation using a Cobalt-60 source in a Gamma Chamber 900 A unit [19]. The samples were stored at room temperature. 2.2. NMR spectroscopy In recent years, ssNMR has emerged as one of the promising techniques to study bone microstructure in a non-invasive manner [20–23]. ssNMR includes two techniques, magic-angle spinning (MAS) and cross-polarization (CP) to improve resolution and sensitivity of NMR active nuclei in the bone sample. Magic-angle spinning reduces line broadening caused by chemical shift anisotropy and dipolar coupling, whereas, cross-polarization improves sensitivity of less sensitivity nuclei such as 13C and 31P by transferring magnetization from abundant 1 H. Intact bones were cylindrically cut (8.0 mm long with a radius of 1.0 mm) so that it could be fixed into a 3.2 mm Zirconium rotor. All

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ssNMR spectra were recorded on a 600 MHz NMR spectrometer (Bruker Biospin, Switzerland) operating at 600 MHz for 1H, 243 MHz for 31P, and 150 MHz for 13C frequencies with a 3.2 mm DVT probe. Magic Angle Spinning (MAS) frequency was set to 10.0 kHz for all experiments controlled by a pneumatic unit with an accuracy of ±2 Hz. The temperature setting of 20 °C was maintained during all experiments. 1H–13C crosspolarization spectra were recorded with a 100 kHz radio-frequency field 1H excitation and decoupling with a sequence with SPINAL-64 [24], 1.0 ms of contact time and 3.0–5.0 s of recycle delays were used during experiments. A total of 10 k and 8 transients were recorded for 1 H–13C CPMAS and 1H spectra respectively. Further, 1H–31P frequencyswitched Lee–Goldberg Heteronuclear Correlation (FSLG HETCOR) experiment was used to selectively observe protons present nearby 31P in the mineral part [21,25]. 50 kHz of effective field was used during 1 H decoupling (FSLG) and 100 kHz 1H decoupling was applied in t2 period respectively. 2D spectra were collected with a typical of 48 data points with 4 transients in the indirect dimension. We determined spin–lattice relaxation time (T1) of 31P from inorganic part by recording inversion recovery experiments with delay time of 2, 250, 700 and 1000 s. Intensities were fitted with single exponential to calculate the inversion recovery time (T1). 2.3. Scanning electron microscopy analysis The surface morphology and pore architecture of bones (normal and osteoporotic) were examined with field emission gun-scanning electron microscopy (FEG-SEM). The bones were cut in small pieces with the help of bone saw diamond cutter and then coated with gold to facilitate the SEM analysis. The morphology of normal and osteoporotic bones was observed by FEG-SEM (JEOL JSM-7600, Japan) with gold/palladium using SC7640 Sputter Coater (Quorum Technologies Ltd, UK). Measurement of elemental composition (Calcium, Phosphorus and Oxygen) was done by energy-dispersive spectroscopy (EDS) and mapping was observed by INCA software (Oxford instruments, Japan). A standard graph of calcium phosphate ratios observed using the EDX system was plotted against the empirical ratio of these standards, calculated from their respective formulae as described by Landis et al. [26]. This graph was used to correct all observed ratios for the osteoporotic and normal bone analyzed in the study. 2.4. Quantification of bone weight loss by thermal gravimetric analysis

between trabeculae, assessed using direct 3D methods) were calculated accordingly [27]. To visualize the 3D representation of the treated defect sites and CTan (Skyscan) were used which further translated into full 3D prototypes using MIMICS® 13.01 (Materialise, Leuvan, Belgium) used at CDRI, India. Images created in the CT analysis were stored in a Dicom format and imported for the 3D model construction. The systematic variation in the bone architecture inside bone construct was analyzed using computer imaging technology, MIMICS® [28]. MIMICS® was used with its automatic segmentation and mesher to reconstruct the geometric model of bone. Further images were analyzed on the basis of number of voxel present in a particular range of Hounsfield Units (HU). Direct correlation between HU unit and apparent density of the bone was classified for elucidation of bone materials. Firstly, bones were scanned and the images stored in a Dicom format. MIMICS® were used to read and reconstruct the geometric model of each type of bone. Using MIMICS® automatic segmentation for the cortical and cancellous bone, different material properties were assigned relating the bone mineral density with the Hounsfield Units (HU). The HU scale is a linear transformation of the linear attenuation coefficient measurement where the radio-density of distilled water at standard pressure and temperature is defined as zero HU and for air at STP is − 1000HU. Bone density is defined as mean value expressed in Hounsfield units in each pixel. In analyzing the pattern and distribution of bone, the threshold employed were 1–225 Hounsfield units (HU) for soft tissue, 226–661 HU for adult spongial bone and 662–1988 HU for adult compact bone as represented by green, red and blue colors respectively [29]. Details of various bone samples are summarized in Table 1. 2.6. Statistics Data are expressed as mean ± SEM unless otherwise indicated. The data obtained in experiments were subjected to one-way ANOVA followed by Tukey's multiple comparison test of significance using GraphPad Prism 3.02 software. Qualitative observations have been represented following assessments made by three individuals blinded to the experiments. 3. Results 3.1. 1H NMR studies to determine variation in water content 1

The amount of degradation of bone samples was quantified by thermal gravimetric analysis (TGA) using NETZSCH-STA 409 PC (Selb, Germany). Samples (n = 3) were loaded within the measurement chamber and heated up to 1000 °C with an ascending rate of 10 °C min−1 in nitrogen environment. The sample chamber was purged with 20 ml/min of nitrogen flow rate. Primary weight change of the samples as a function of temperature was recorded.

H NMR spectra of various normal and osteoporosis bone samples after different processing steps are shown in Fig. 1. All 1H NMR spectra from different sets of samples and processing steps were recorded and processed under identical conditions. We used peak shapes and intensities as a quantitative measure of bone quality. All peaks were externally referenced to 1H NMR spectra of water at 4.7 ppm.1H NMR spectra of all bone samples showed two

2.5. Analysis of bone using μCT and MIMICS®

Table 1 Nomenclature used for bone samples.

The bones were analyzed with a commercially available desktop μCT 1172 scanner (Skyscan, Kontich, Belgium). Scans were obtained at 100 kV and 100 μA with the use of an aluminum–copper filter to optimize the contrast, a 360° rotation, an average of four frames and a rotation step of 0.4o (2700 images per scan). The reconstruction software (NRecon v.1.4.4) was used to create 2D 2000 × 2000 pixel images. ROI of cortical and cancellous bone from each sample was then applied for the analysis of 3D micro-architecture parameters using CTAn software (Skyscan, Kontich, Belgium). Trabecular bone volume (BV/TV; percentage) (ratio of the segmented bone volume to the total volume of the region of interest), trabecular number (Tb.N, 1/mm) (measure of the average number of trabeculae per unit length), trabecular separation (Tb.Sp; mm) (mean thickness of trabeculae, assessed using direct 3D methods) and trabecular thickness (Tb.Th; mm) (mean distance

(A) Types of bone Type of bone

Normal

Osteoporotic

Cortical Cancellous

NCo NCa

OCo OCa

(B) Processing step details S. no. Step no. Code used Short name of process Process details 1 2 3 4 5

1 2 3 4 5

U P A FD R

Unprocessed Pasteurized Alcohol Freeze dried Radiated

6

1–5

F

Full process

Normal washed bone Pasteurized at 60 °C for 3 h Placed in 70% ethanol for 1 h Stored at −80 °C Irradiated using 25 kGy of gamma radiation Step 1 to Step 5 complete

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Fig. 1. 1H ssNMR MAS spectra at MAS frequency of 10 kHz for normal and osteoporotic bones showing profile of bound water and lipid/OH− in various bone samples and changes during different processing steps. (A) 1H ssNMR MAS spectra of normal cancellous bone; (B) 1H ssNMR MAS spectra of all unprocessed bone; (C) 1H ssNMR MAS spectra of all full processed bone; (NCo—Normal Cortical; OCo—Osteo Cortical; NCa—Normal Cancellous; OCa—Osteo Cancellous; U—Unprocessed; P—Pasteurized; A—Alcohol; FD: Freeze Dried, R—Radiated; F—Full Process).

peaks, one peak at ~5.2 ppm originating from water in bone matrix and another at 1.4 ppm which was assigned to hydroxyl ion (OH−) attached to Ca2+/organic matrix as per previously reported values [21,23]. When we compared proton spectra for the same set of samples but processed in a different manner, we found that both resonances got affected differently (Fig. 1A). Although in all processing steps, water appeared to be intact in the bone matrix showing a broader peak, however, water behavior remarkably changed in case of the pasteurized sample. Water line was sharper, indicating that pasteurization could affect the local environment of bound water. A progressive increase in the 1.4 ppm peak, Ca2+/organic matrix was observed with processing steps [22]. Alcohol treatment and freeze drying methods appeared to affect the overall profile of this peak as only one resonance was observed in the spectra. A significant change was observed for the bone undergoing complete processing steps where water to lipid/hydroxyl ratio had improved significantly (Fig. 1A and Supporting Information Figure S1). Proton spectra for osteoporotic samples showed remarkably different features for water and hydroxyl peaks (Fig. 1B and C). In osteoporotic cortical bone, the water line was sharp, whereas in cancellous bone, the water peak intensity was less, suggesting that in the osteoporotic cases, either water was not bound to the bone matrix or the amount of water diminished significantly. Both of these cases clearly indicated impaired water microstructure for osteoporotic samples. Further, we recorded proton spectra for each of the processing step for both osteoporotic samples. We noticed that water quality gradually improved and after complete processing the water appeared to be more intact in the bone matrix. Similarly, water/hydroxyl intensity ratio also improves for the full processed samples. In Supporting Information Figure S1, we compared normal cortical/cancellous with their osteoporotic counterpart. In case of cortical bones, the water line was significantly sharper than osteoporotic bone samples suggesting that the water could be entrapped in the pore, or in the collagen matrix which is associated with water. A significant improvement in the line shape as well as the ratio was observed for fully processed samples. On the other hand, for the cancellous bone, water peak intensity in osteoporotic trabecular

bones was low. Similarly, for trabecular bones, a remarkable improvement in the quality was observed when samples were fully processed. 3.2. 13C NMR studies to assess collagen structure The structural changes in the organic component of bone by 13C CPMAS spectra were also analyzed. Bone matrix has type 1 collagen as its organic component and 13C spectra provide a structural and integrity signature for the bone (Fig. 2). Many well resolved signals were obtained in the 13C CPMAS spectrum of bone that can be assigned to the backbone and side chain of the major amino acids in collagen type I. A resonance at 17.6 ppm for Ala Cβ and Hyp Cγ peak at 71.1 ppm confirmed that collagen was in a triple helical state; similarly, the appearance of three well-resolved peaks in the carbonyl carbon chemical shift region (∼170 ppm) indicated the native collagen structure in the unprocessed bone samples, which was in agreement with a previous study [21,23]. Resonances for the unprocessed cortical bone samples were sharper and better resolved than their cancellous counterpart suggesting that collagen was structurally more ordered in these cases. Moreover, relative intensities for different resonance were similar. Various processing steps adopted in our protocol did not appear to change the overall collagen structure. Further, we used Gly Cα peak to monitor relative shift in the resonance frequencies of various atoms under different processing steps. Many peaks including the Hyp Cγ, Hyp Cβ, Pro Cα, Pro Cβ, and Pro Cγ showed shift in peak positions (Supporting Information Figure S2). For example, pasteurized samples showed downfield (increased chemical) shift that eventually returned to its original value in the fully processed sample. 3.3. 1H–31P HetCor and T1 studies to determine local structure and integrity of bone samples Subsequently, we performed 1H–31P hetero-nuclear correlation (HetCor) experiments to obtain microstructure of the inorganic component of the bone matrix. HetCor uses dipolar couplings between 1H and

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Fig. 2. 1H decoupled cross-polarized magic angle spinning (CPMAS) 13C ssNMR spectra of normal and osteoporotic bones recorded at 20 °C and a MAS frequency of 10 kHz showing various amino acid components of collagen I. The peak assignment was taken from the literature [23]. (A) 13C ssNMR spectra of normal cancellous bone; (B) unprocessed bone; (C) full processed bone; (NCo—Normal Cortical; OCo—Osteo Cortical; NCa—Normal Cancellous; OCa—Osteo Cancellous; U—Unprocessed; P—Pasteurized; A—Alcohol; FD: Freeze Dried, R—Radiated; F—Full Process).

31

P nuclei, which are dependent on the distance between coupled nuclei (1/r3) to probe 1H structural arrangement in the vicinity of 31P atom at an atomic level. Here, one could selectively excite 1H signal associated with water or the organic component of the bone and monitor the spatial arrangement of the inorganic component of the bone. Although 1 H–31P HetCor is not quantitative, it is possible to assess relative bound water content with respect to structural OH− [20]. HetCor spectra showed more than two well-resolved peaks; the peak centered at 0.2 ppm was assigned as (OH−) and the peak at ~5.2 ppm corresponded to the water bound to inorganic matrix (Fig. 3A-F).1H–31P HETCOR spectra of various bone samples are shown in Fig. 3A-F. Interestingly, we observed a third peak at ~ 7 ppm that could be assigned to HPO4 − as reported earlier [30]. Appearance of such a peak suggested that the apatite matrix in human bone is quite close to organic and water components. Next, we probed the quality of inorganic component of bone by measuring 31P longitudinal relaxation (T1) parameters. T1 parameter can provide information about the strength of bone and crystallinity of the inorganic matrix [20]. In this study, because normal and osteoporotic bone samples had different structural hydroxyl group (OH−) therefore T1 parameter was expected to be different. T1 relaxation for 31P is mediated via 1H–31P dipolar mechanism; therefore, local environmental change due to processing steps will have effect on the measured T1 values. Additionally T1 showed strong correlation with classical parameters used to access bone microstructure such as bone volume/trabecular volume (BV/TV), trabecular thickness (Tb Th), trabecular number (Tb N) and trabecular separation (Tb.Sp) in rat bones [20]. Fig. 3G shows the measured T1 values for different samples and changes due to various processing protocols. Normal and osteoporotic cortical bones have larger T1 than their cancellous counterparts suggesting that cortical bone strength and crystalline order was significantly higher than the cancellous bone.

Osteoporotic samples were less ordered and therefore these samples had lower T1 values. All individual processing steps appeared to affect the crystalline order of bones. However, pasteurization and radiation showed significantly lower T1 values indicating that these two steps could change the inorganic matrix of the bone. For the fully processed sample, T1 values were comparable to the unprocessed counterparts suggesting that the full processing restored the crystalline quality and strength of bone allografts.

3.4. SEM analysis provided insight into bone morphology SEM is a microscopy technique that is used to probe the microstructure of bones. The Ca/P ratio can be quantified, which accounts for the bone quality, while the mapping reveals the distribution of elements throughout the bone samples. At a macroscopic level, it describes progressive color changes, fracturing and distortion processes in the bone. In this study, SEM showed differences in the micro-structural properties and the distribution of various elements in the inorganic matrix of both cortical and cancellous bone. Normal bones had continuity in the crystal structure whereas the osteoporotic bone mineral exhibited discontinuous crystal arrangement suggesting defects in the lattice as shown in Fig. 4. At low magnification, SEM images of osteoporotic bone showed trabecular structural degradation, with cavities due to excessive resorption of the trabecular arches. The osteoporotic bones were marked by trabeculae with thin and flat walls having numerous cracks. In normal bone, the trabeculae were intact with interconnected thick-walled arches and rare instances of trabecular cracks. The SEM images of the osteoporotic bone clearly indicated low mineral density and appeared like a spongy structure that would impart fragility to the bone. Surface SEM views of normal and osteoporotic bone (Supporting Information

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Fig. 3. (A) 2D Heteronuclear (1H–31P) experiments at MAS frequency of 10 kHz of different bone samples (contact time CT = 1 ms) (A) NCo-U (green), NCo-F (blue); (B) OCa-U (green), OCa-F (blue); (C) NCo-U (green), NCo-F (blue) NCo-R (red); (D) NCa-F (blue) &OCa-F (green); (E) NCo-F (green) & OCo-F (blue) and (F) ratio of lipid (OH−) and water; (G) T1 measurement of cortical and cancellous bone samples showing the rigidity of bone; (H) Ca/P ratio showing the quality of bone samples (n = 3 samples from each group).

Figure S3) shows dense surfaces for normal cortical human bone and a few pores of 38 μm average size for osteoporotic cortical human bones. EDX was used to compare the calcium to phosphorus (Ca/P) molar ratios in normal and osteoporotic bone in accordance with standards of known composition. The Ca/P ratio values for normal bone was 1.67 that correlated well with chemically prepared hydroxyapatite (Ca/P = 1.60), whereas osteoporotic bones had significantly lower Ca/P ratio (1.49) which correlated well with previously published values [31] (Fig. 3H). These values suggested that the carbanoapatite usually found in bone appears to be disturbed in osteoporotic samples. Carbanoapatite is considered to be the main mineral component of normal cortical bone, the presence of a mineral with lower molar Ca/P ratio in osteoporotic bone suggested that the mineral was altered from the normal carbanoapatite. This could attribute to the increased fracture rate observed in osteoporosis. EDX results showed that Ca/P ratio was not significantly different between unprocessed and other processing groups within the same group. However, there was a significant difference (P b 0.05) between normal and osteoporotic cortical bone. In contrast to this, cancellous bone showed a significant difference in Ca/P ratio between normal and osteoporotic bone. SEM-mapping analysis demonstrated differences in the distribution patterns of atoms. Ca and P had similar atomic mapping patterns in all cases whereas oxygen seemed to be clustered in osteoporotic samples. Normal cortical bones had uniform distribution of all elements that appeared to have significantly perturbed in the osteoporotic cancellous bones.

SEM results only provide bone structure in two dimensions, necessitating the use of a technique that could be used for investigating threedimensional architectural analysis. High-resolution 3D imaging μ-CT techniques was used for evaluation of bone morphology, microarchitecture and analysis of the three-dimensional trabecular bone. Mineralization of bone is responsible for its hardness and organic component accounts for hard and soft areas in the bone. Collectively these two components together with structural water are accountable for bone unique properties. NMR and SEM cannot by themselves give bone hardness values, which μCT, together with MIMICS® analysis of the X-ray voxel data can provide. Here for the first time we explored these unique features. 3.5. μ-CT and MIMICS® analysis to access the bone micro-architecture Three-dimensional μCT evaluation of normal and osteoporotic bones trabecular microarchitecture of the different groups with or without processing showed that the percent bone volume (BV/TV; %) Tb.N (1/mm), Tb.Th (mm) and Tb.Sp (mm) were comparable among all groups of the normal bone samples (Fig. 5). However, all these parameters exhibited marked reduction (b0.001) in all groups of osteoporotic bone samples when compared with normal bones. Moreover, pasteurization appeared a punitive processing for the osteoporotic bone samples because BV/TV, Tb.N both showed marked reduction (~20%) and increase in Tb.Sp (~20%) when compared to unprocessed osteoporotic bone group, while the values of these

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Fig. 4. SEM micrographs of normal and osteoporotic bone sample: Normal bone showing continuity in crystal structure, whereas osteoporotic bone showing discontinuity in the crystal structure. Rows represented as: 1st row: NCo—normal cortical bone; 2nd row: OCo—osteoporotic cortical; 3rd row: normal cancellous; 4th row: osteoporotic cancellous. Columns represented as: 1st column: low magnification at 100×/50×; 2nd column: medium magnification at 500×; 3rd column: high magnification at 10,000× and 4th column: elemental mapping [distribution of elemental composition (Calcium: green, Phosphorus: blue and Oxygen: red) as shown by SEM-Mapping].

parameters in other processing groups were comparable (Fig. 5). Pasteurization significantly attenuates the trabecular micro-architecture of the osteoporotic bone samples which is in accordance with our T1 (sec) and Ca/P (molar ratio) data (Fig. 3G and H). The discretization for the HU computed by MIMICS® is presented in Fig. 6. Our analysis showed highest density of number of voxel in the range of 661 to 1980 in the cortical case, whereas, for cancellous bone voxel distribution had a wider spread in the region of 1–1600. This indicated a high bone density in cortical than the cancellous counterpart. In osteoporotic cortical and cancellous bones, number of voxels was reduced by 20% in comparison to normal bones (Supporting Information Table 1). In each case, processing steps shifted HU values whereas full processing preserved similar HU value as compared to the unprocessed bone. Pasteurization and alcohol treatment affected soft materials in the bone and therefore, HU value shifted toward the higher unit. On the other hand, radiation treatment appeared to shift HU values toward higher units, suggesting that gamma ray radiation could perturb bound water integrity, which in turn, could make bone weaker. Here again, complete processing restored all parameters that might have been perturbed in the individual treatment.

started at 1000 °C [32]. Final residual mass (50–60%) remaining in normal bone samples corresponded of hydroxyapatite. The average amount of water and organic materials lost during TGA measurements were calculated and summarized in Table 2. Approximately 5–8% and 8–11% of water loss was observed in the temperature range of RT to 160 °C for normal bones and osteoporotic bones respectively. Further, 23–25% and 23–28% of organic constituents were removed in normal bone and osteoporotic bone samples between 200 °C and 600 °C. Around 5.5–6.5% of weight loss was measured in both types of bones from 600 °C to 1000 °C. We noticed that freeze drying methods affected water and organic constituents significantly higher in comparison to other processing methods in the osteoporotic bones. 3.6.1. Degradation profile divided in to three categories 1. 30–160 °C — Amount of water loss; 2. 200–600 °C — Amount of Protein loss; 3. 600–1000 °C — Amount of Residual protein, organic substance and water loss. 4. Discussion

3.6. Thermal stability and degradation pattern of bone TGA was used to analyze thermal stability of bone samples that provide the distinct degradation pattern in various phases. The first weight loss was observed from room temperature (RT) to 160 °C, which corresponded to the removal of bound water from collagen matrix. The second weight loss appeared between 200 °C to about 600 °C that was associated with the loss of collagen, lipids and other proteins. The third weight loss weight represented the removal of water from the inorganic matrix and remaining organic components in the range of 600 °C to 850 °C. Finally, mineral phase (calcium phosphate) loss was

We have used a variety of techniques to characterize micro- and macro-structural features of bone in normal and osteoporotic bone. Zhu et al. [23]reported insignificant variation in peak at 1.4 ppm after dehydration for intact bone and suggested that this peak could be used as an internal reference for determining relative changes in the water content [23]. Therefore, we used peak at 1.4 ppm as internal reference to access relative change in water content. To determine the ratio of total water content with respect to OH−, we integrated water peak (8.0–3.5 ppm) with respect to OH− resonance (3.0–0.5 ppm). We found that ratio of water to OH− balance improved for fully

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Fig. 5. μ-CT analysis of different bone samples: parameters of cancellous bone. (A) Representative three-dimensional μCT images showing changes in trabecular microarchitecture. (B–D) Showing the quantification data of respective 3D images; (B) BV/TV (%), (C) Tb.N (1/mm), (D) Tb.Th (mm), (E) Tb.Sp (mm); values are expressed as mean ± SEM (n = 3 samples per group); * p b 0.05 vs. U was determined by one way ANOVA followed by Tukey's multiple comparison test. With respect to normal bone all parameters in osteoporotic bone samples have p value b0.001.

processed normal and osteoporotic bones (Supporting Information Figure S1). It was shown that grinding of bone changed the water content as well as homogeneity of the bone matrix [23], hence intact bones were used in this study. Water content and integrity was verified by recording 1H NMR spectra and monitoring line shapes and intensities of the proton spectra. We observed that the water content was almost the same even after seven days of MAS experiments. Subsequently, water in different types of intact bone and the effect of various preservation protocols on the quality of the bone was probed by proton spectra. For osteoporotic bone samples, water resonances showed a high degree of alteration with a very sharp line indicating free water that could have

been trapped in the extracellular matrix or in the pore of the bone, whereas, for the osteoporotic cancellous sample, the quantity of water was extremely low. Both these results indicated that in the case of fragile osteoporotic bone, water quality and quantity were highly imbalanced. Various processing steps adopted for bone preservation could also affect the bone quality. Preserving bone in 70% ethanol or radiating bone with gamma ray to avoid disease transmission were shown to affect the water microstructure. Many researchers have investigated the effect of freeze drying and ethanol treatment on cortical bovine or goat bone. These studies concluded that the above-mentioned processing steps significantly affected water content. Dehydration was reported to bring the collagen and hydroxyapatite closer which results into

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Fig. 6. Mimics analysis: Combined effects of CTan and Mimics on (A) normal cortical bone, (B) osteoporotic cortical bone, (C) normal cancellous bone, (D) osteoporotic cancellous bone. In analyzing the pattern and distribution, the threshold employed were 1–225 Hounsfield units (HU) for soft tissue, 148–661 HU for spongial bone and 662–1988 HU for compact bone as represented by green, red and blue color respectively.

fragility of the bone [21,33]. In the case of osteoporotic bone or dehydrated bone ductility of femur bone decreases and bone become prone to brittleness [34]. In the MIMICS® analysis, osteoporotic samples have lower number of voxels in the range of 662–1988 and 226–661 HU values indicating that these bones have reduced bound water and therefore their mechanical strength was compromised. However, complete processing as suggested in our study, appeared to preserve the water quality, as indicated by the ratio of water to lipid/OH− peak for all the allografts.

Table 2 Quantification of weight loss (in %) of bone as heated from room temperature to 1000 °C. Temp °C

NCo-U

NCo-P

NCo-A

NCo-FD

NCo-R

NCo-F

30–160 °C 200–600 °C 600–850 °C 600–1000 °C

5.302 24.357 5.022 6.07

6.83 23.626 3.5 6.084

6.637 24.736 3.565 5.943

6.941 25.294 5.038 5.833

7.693 23.062 3.577 5.945

7.11646 22.46745 3.57035 5.74185

Temp °C

OCo-U

OCo-P

OCo-A

OCo-FD

OCo-R

OCo-F

30–160 °C 200–600 °C 600–850 °C 600–1000 °C

8.883 23.39 3.636 6.244

10.914 28.317 3.255 6.216

9.619 27.335 3.457 6.323

10.551 20.666 3.135 5.797

7.941 27.199 4.427 6.4

9.305 23.111 3.567 6.235

The organic component of bone matrix consists of citrate, other lipids (b 5%) and polysaccharides along with various other non-collagenous proteins (b 5%), and type I collagen 90% [21,34]. In natural abundance 13C NMR spectrum, mostly peaks come from the type I collagen fiber. Therefore, one dimensional 13C spectrum is a reliable reporter for the collagen structure in bone matrix. Many studies have shown that dehydration of bone grossly affected the collagen structure as line became broader and few of the resonances disappeared from the spectrum [20,23]. However, in this study no significant perturbation in the structure of collagen was observed. All peaks appeared to be intact as expected for well-preserved collagen triple helix structure. In cancellous bone, the structure and intactness of collagen appeared to be slightly perturbed as indicated by relatively broader resonances (Fig. 2B and C). Such a phenomenon can be explained as follows: Various processing steps are bound to change the collagen structure depending upon the extent of their harshness. For example, alcohol treatment or dehydration can significantly perturb the triple helix characteristics of collagen I. Processing steps adopted in this study are mild and a combination of all the processing steps helped in regaining loss in structural features of collagen I as observed in the 13C spectra. Among all NMR parameter 31P T1 directly reports the strength and micro-structural properties such as BMD, BV/TV and Tb.N., on structural OH− and mineral crystallinity [20]of the bone matrix [20,21]. It was

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shown that T1 was also reduced in the experimentally generated osteoporotic rat bone. Here, 31 P T1 values were higher for cortical bone than for cancellous bone. Similarly, T1 values of normal bones were higher than that of their osteoporotic counterparts. As expected, cortical bones have better ordered structure and are more crystalline than cancellous bone, and osteoporosis not only affects water composition but also drastically reduces bone mineral. Freeze-drying and alcohol treatment did not affect inorganic matrix in any sample. However, radiation and pasteurization considerably reduced T1 values. Water interaction with organic and inorganic component is a crucial parameter for bone quality and stability. Bound water, which is present in the extracellular matrix of bone, forms a water-bridge between inorganic Ca2+ and PO4− and with side chains of collagen fiber [35]. A quantification of bound water with respect to OH− present in the bone can report about porosity and strength of the bone. In 31P dimensions, we observed the peak at ~ 4 ppm corresponding to phosphorus of the bone matrix. Unlike previously published reports, we noticed three peaks in the proton dimension, namely bound water (~5.0 ppm), hydroxyl (0.2 ppm) and HPO4− (~7.0 ppm). Appearance of HPO4− peak in our sample clearly indicated that human bones are intricately connected with the collagen matrix. Such an interaction could explain the strength of human bone originating from hydrogen bonding of structural water molecules [30]. Bone undergoes continuous remodeling throughout life under the influence of hormonal and physical factors. Small quantities of bone minerals are constantly lost in a process called resorption, and simultaneously new minerals are deposited. In adults, these processes must be balanced if bone strength has to be maintained. In case of more mineral loss than deposition, bone weakens; and can become osteoporotic or brittle. The remodeling and restructuring of bone happens throughout the life. During aging bone becomes thicker and less dense because of loss of trabecular bone, widening of the bone cavity, and thickening of cortical layer [36]. The mechanical properties of bone are determined by the micro-architecture of the trabeculae, comprising their number and thickness, and the presence of cavities. Gonadal hormones maintain the skeleton during the reproductive years of life and their deficiency due to aging is a major cause of bone loss. Women in particular are greater affected than men in terms of bone loss as estrogen, the strongest osteoprotective hormone ceases to secrete due to menopause which results in osteoporosis and consequent increase in fracture risk [37]. An imbalance of RANK/RANKL/osteoprotegerin in the signaling pathway for osteoclasts and osteoblasts can be one of the causes for osteoporosis [38,39]. Previous animal studies showed the imbalance in osteoprotegerin, which leads to an increase in bone mass and loss in protein, leads to osteoporosis and increased fracture risk [40]. Therefore, inhibitors of RANKL emerge as potential treatment for osteoporosis in humans [38,39]. Mechanical loading provides bone forming stimulation and thus physical exercise is a recommended non-pharmacological approach to protect against post-menopausal bone loss. The microstructure of osteoporotic bone demonstrates a reduced amount of bone mineral and thinning of the bone walls along with presence of cracks and perforations, resulting in loss of integrity of the trabecular arches [41]. SEM images clearly showed a demarcation in all these parameters for the normal and diseased bone. In EDX analysis, low Ca/P molar ratio, was observed for osteoporotic samples. This could be due to an increase in lattice protons, decrease in lattice OH−, and decrease in lattice carbonate. There could be several explanations for reduced Ca/P ratio for osteoporotic bone: 1. Excess HPO− 4 groups were adsorbed on the surface of apatite crystals, 2. Calcium ions were removed from the surface or from the interior of the crystals. It could also be possible that calcium ions would be replaced by hydronium ions to preserve neutrality in the bone. The consequence of all these imbalances was responsible for reduced Ca/P molar ratio, which in turn can cause brittleness of the bone. The well-resolved peaks (Ca, P, O) observed in the normal case

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indicated the crystallinity of the healthy bones, whereas in osteoporotic the crystallinity was reduced. The decrease in crystalline order indicated the weak structure of the inorganic phase which imparts weakness and brittleness of the osteoporotic bone. TGA analysis confirmed that normal bone was even thermally stable, with reduction in the content of its hydroxyapatite; stability also reduces in osteoporotic case. This effect is not only due to ordered structure of the hydroxyapatite but also because of an interaction of collagen and water in an appropriate fashion. TGA showed that in osteoporotic bone the extent of water evaporation and collagen degradation was high. Reduction of the hydroxide ion from the apatite crystals decreases its stability at higher temperature. These results were in good agreement with previous findings [42], where organic components such as collagen, fat tissues and proteins were removed as the samples were heated up to 200 °C and completed at approximately 600 °C. Catanese et al. [43] reported that around 85% of the organic content was removed upon heating the bone up to 350 °C. A remarkable cross-correlation was observed in the various bone integrity parameters such as T1, Ca/P ratio and MIMICS® analysis. Higher T1 value which is an indicator of rigid structure is also substantiated by higher Ca/P ratio and number of voxels of MIMICS® analysis. The potential of combining μ-CT with MIMICS® was exploited in the study to monitor the effect of processing on normal and osteoporotic bone. Porosity, surface area per unit volume, and the degree of interconnectivity were evaluated through imaging and further by computer aided manipulation of the bone μCT scan data. Such an analysis provided in-depth micro-architectural arrangement in the bone. 5. Conclusion Taken together, this study investigated a wide range of normal and osteoporotic bone samples that are used as allografts. By using a variety of biophysical and microscopy techniques, it was observed that the micro-structural properties of cortical and cancellous bone in the normal and osteoporotic states vary significantly. Intricate balances between water, organic component and inorganic component can be greatly changed depending on the way the bone was processed and preserved. The preservation protocol suggested herein restored all critical parameters, which was cross-validated by different techniques. Detailed structural parameters reported in the present study could also be helpful in designing an appropriate cost-effective bone-scaffold. The processing protocol described here, restored important parameters and is therefore recommended as a means of preserving normal as well as osteoporotic bone for use as allografts in bone reconstruction. Such an outcome will be implicated in providing critical micro-structural parameters for BA by which non-human (xeno or synthetic bone graft) can also be evaluated. Authors' roles Study design: AKS, AK, ALG, NS and JRB. Data Recording: AKS, RKR, MPK, CS and TB. Data analysis: AKS, RKR, MPK, JRB, NS and AK. Data interpretation: AKS, NS, SV, AK and JRB. Drafting manuscript: AKS, ALG, NC, NS, AK and JRB. Approving final version of manuscript: AKS, ALG, NC, NS, AK and JRB. Acknowledgments The authors are grateful for the technical assistance provided by Ms. Urmila Samant and Ms. Cynthia D'Lima at the Tissue Bank, TMH, Mumbai, for processing the human bone samples according to the various protocols. The authors are also thankful to Sophisticated Analytical Instrument Facility (SAIF) and Centre for Research in Nanotechnology and Science (CRNTS), Indian Institute of Technology Bombay, Mumbai for the SEM and TGA Facility. We thank Rajan Singh, Nitin Sagar, Mayur Temgire and Rohit Teotia for helping in characterization. Atul

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Kumar Singh is supported by a fellowship DST-INSPIRE from the Department of Science & Technology (DST), Government of India. NC acknowledges funding from CSIR (BSC0201). NS acknowledge funding from DBT, India (BT/PR12700/BRB/10/719/2009). AK acknowledges funding from DST, India (SR/SO/BB/022/2012) and CSIR, India (37(1509)/11/ EMRII). JRB acknowledges funding from DBT, India (BT/PR3138/NNT/ 28/550/2011). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.msec.2016.02.002. References [1] S.V. Kothiwale, P. Anuroopa, A.L. Gajiwala, A clinical and radiological evaluation of DFDBA with amniotic membrane versus bovine derived xenograft with amniotic membrane in human periodontal grade II furcation defects, Cell Tissue Bank. 10 (2009) 317–326. [2] M. Shah, A.L. Gajiwala, S. Shah, D. Dave, Comparative study of indigenously prepared and imported, demineralized, freeze-dried, irradiated bone allograft in the treatment of periodontal infrabony defects, Cell Tissue Bank. (2014) 1–9. [3] C. Centers for Disease, Prevention, hepatitis C virus transmission from an antibodynegative organ and tissue donor—UNITED States, 2000–2002, MMWR Morb. Mortal. Wkly Rep. 52 (2003) 273. [4] L. Ireland, D. Spelman, Bacterial contamination of tissue allografts experiences of the donor tissue bank of Victoria, Cell Tissue Bank. 6 (2005) 181–189. [5] T. Eastlund, M.K. Winters, Testing the Tissue and the Environment, Essentials of Tissue Banking, Springer, 2010 167–187. [6] A.L. Gajiwala, M. Agarwal, A. Puri, C. D'Lima, A. Duggal, The use of irradiated allografts in reconstruction of tumor defects—the Tata Memorial Hospital experience, Cell Tissue Bank. 4 (2003) 125–132. [7] O. Cornu, X. Banse, P.L. Docquier, S. Luyckx, C.H. Delloye, Effect of freeze drying and gamma irradiation on the mechanical properties of human cancellous bone, J. Orthop. Res. 18 (2000) 426–431. [8] H. Nguyen, D.A.F. Morgan, M.R. Forwood, Sterilization of allograft bone: effects of gamma irradiation on allograft biology and biomechanics, Cell Tissue Bank. 8 (2007) 93–105. [9] P. Janicki, G. Schmidmaier, What should be the characteristics of the ideal bone graft substitute? Combining scaffolds with growth factors and/or stem cells, Injury 42 (2011) S77–S81. [10] A. Oryan, S. Alidadi, A. Moshiri, N. Maffulli, Bone regenerative medicine: classic options, novel strategies, and future directions, J. Orthop. Surg. Res. 9 (2014) 18. [11] World Health Organization, Guidelines on viral inactivation and removal procedures intended to assure the viral safety of human blood plasma products, WHO Technical Report, Series No. 924, 2004. [12] S. Zoricic, D. Bobinac, B. Lah, I. Maric, O. Cvijanovic, S. Bajek, V. Golubovic, R. Mihelic, Study of the healing process after transplantation of pasteurized bone grafts in rabbits, Acta Med. Okayama 56 (2002) 121–128. [13] W.A. Rutala, D.J. Weber, C. Centers for Disease, Guideline for DISINFECTION and sterilization in HEALTHCARE facilities, 2008, Centers for Disease Control (US), 2008. [14] C. Gardin, S. Ricci, L. Ferroni, R. Guazzo, L. Sbricoli, G. De Benedictis, L. Finotti, M. Isola, E. Bressan, B. Zavan, Decellularization and delipidation protocols of bovine bone and pericardium for bone grafting and guided bone regeneration procedures, PLoS One 10 (2015), e0132344. [15] M.F. Moreau, Y. Gallois, M.F. Baslé, D. Chappard, Gamma irradiation of human bone allografts alters medullary lipids and releases toxic compounds for osteoblast-like cells, Biomaterials 21 (2000) 369–376. [16] H. Nguyen, D.A.F. Morgan, M.R. Forwood, Validation of 11 kGy as a radiation sterilization dose for frozen bone allografts, J. Arthroplast. 26 (2011) 303–308. [17] K.C. McGilvray, B.G. Santoni, A.S. Turner, S. Bogdansky, D.L. Wheeler, C.M. Puttlitz, Effects of (60)Co gamma radiation dose on initial structural biomechanical properties of ovine bone–patellar tendon–bone allografts, Cell Tissue Bank. 12 (2011) 89–98.

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