Introduction

Fish as a diet has several nutritional and therapeutic benefits for health. The most important constituents of fish as food are protein, fat, carbohydrates, sodium, potassium, calcium, magnesium, vitamin B6 and vitamin B12, which help in maintaining good health as well as the central nervous system of a body [1]. Apart from its nutritional values, fish waste like scale, fin and bone is an important source of hydroxyapatite (HAp) and collagen biomaterials. Each scale consists of two distinct regions: an external (osseous) layer and an internal fibrillary plate. In the upper external layer, collagen fibers are randomly arranged and embedded in a proteoglycan matrix. The collagen fibers are produced within the fibrillary layer by scleroblasts located at the base of the scales. Mineralization of the scales occurs continuously throughout the life of the organism. The external layer is initially mineralized with matrix vesicles and calcium phosphate-based materials are deposited biologically on the layer matrix. HAp is the most stable calcium phosphate salt at normal temperatures with a pH between 4 and 12 [2]. It is an important material of great interest in protein chromatography applications, waste water treatment processes and suitable scaffold materials [3, 4]. HAp is also considered as a model compound to study biomineralization phenomena [5, 6]. There is an intensive attempt for the development of well-defined HAp crystals toward physicochemical behavior in vitro and in vivo studies for reconstructive bone replacement and other medical applications [7, 8]. In literature, several methods of preparation of HAp crystals from bio-wastes have been reported, including solid-state reactions, crystal growth under hydrothermal reaction, layer hydrolysis of other calcium phosphate salts, sol–gel crystallization [9–15]. Due to low production cost and worldwide availability, the researchers [16] are concentrated on the use of fish scale bio-waste for the processing of HAp crystal [17]. Many processing technologies have been employed to obtain porous ceramics for bone tissue engineering from different bio-wastes. In tissue repair application, the macropores and highly interconnected networks are required for the growth of surrounding host tissues. The porosity of filler scaffold is about 30–33 % which is also similar to the results reported by Gross et al. [18]. The optimal scaffold design and fabrication techniques must be able to create porous structures adequate for attaining the desired mechanical function and mass transport properties [18, 19]. In this paper, an attempt has been made to synthesize HAp powder from Labeo rohita scale. The powder is characterized through thermogravimetric analysis, X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR), etc. The mechanical properties of the sintered HAp have been evaluated for development of porous scaffold. The cell attachment on HAp surfaces, cytotoxicity evaluation and MTT assay on RAW macrophage cell line media are carried out to demonstrate good biocompatibility.

Methods and materials

Synthesis and characterization of hydroxyapatite powder from fish scale

Labeo rohita scales are used as raw material for HAp processing. Scales are initially washed thoroughly with water to discard impurities and then deproteinized by solvent system. The cleaned de-protienated scale is calcined at 700–800 °C to synthesis HAp ceramics after differential thermal analysis of scale from room temperature to 1,200 °C by NETZSCH Jupiter STA 491 at a heating rate of 10 °C/min. XRD Analysis of HAp powder is performed at the scanning range of 2θ = 20°–80° with CuKα target by Shimadzu, XRD-6000 instrument. The crystallographic phases of HAp with different temperatures are recorded and confirmed the HAp using standard JCPDS files. FTIR analysis is carried out at the scanning range of 4,000–400 cm−1 (Shimadzu IR Prestige—21, Japan) to determine functional groups present in HAp powder. FESEM and EDX (Model No: SUPRA 40, CARLZEISSSMT, Oxford) analyses reveal the compositional and morphological structure of green as well as sintered powder of HAp. Quantitative energy dispersive X-ray is also performed to confirm the Ca/P ratio of the sample apart from chemical method. Ultrasonic instruments are employed for dispersing and homogeneously distribute the particles into liquids to prepare samples for TEM analysis (TEM, Jeol Gem Microscope). “Image J” software is used to evaluate the typical images of HAp particles.

Preparation of fillers and scaffold

The calcined powder is wet ball-milled for 48 h in planetary mill. The particle size of dried milled powder is measured in Malvern particle size analyzer. The average size of the particle is ~0.5 μm. Triton–X surfactant is added drop wise to the dried ball-milled powders to make a paste for injection molding. The sample is prepared as small rod-shaped fillers with 2 mm diameter and 4 mm long. The fillers are then dried at 80 °C for several hours. Finally, the dried samples are sintered at 1,200 °C for 2 h. Alternatively, hydroxyapatite samples are mixed well with requisite amount of starch porogen and compacted with unidirectional pressure at 200–250 MPa to make 12 mm dia. The sample was sintered at 1,200 °C for crystallographic phase determination as well as physico-mechanical property evaluation. The process flow of fish scale-derived HAp scaffold development and in vivo bio-implantation is shown in Fig. 1.

Fig. 1
figure 1

Pictorial representation of a fish Labeo rohita, b chemically treated dried fish scale, c scanning electron microscopy of treated fish scale, d synthesized HAp powder, e fabrication of small fillers by injection press molding, f sintered fillers, g SEM images of porous fillers, h schematic representation of fillers in rat model

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Results and discussion

Differential thermogravimetric analysis

The DTA/TG analysis reveals the phase transformation and determines the weight loss with respect to temperatures. Two major and one minor mass losses are distinctly observed as depicted in Fig. 2. The first major weight loss is observed in the range of 100–400 °C, on the heating process from room temperature to 1,200 °C. This mass loss is reported as near about 4.56 % of the sample taken for experimentation. The first mass loss process may be due to the removal of adsorbed water from the surface and inter-particle spaces [17]. Other exothermic peaks at ~407 and 709 °C correspond to weight losses about 0.86 and 0.17 %, respectively. The weight loss at the range of 400–500 °C is due to the combustion of organic materials. However, there is a minor weight loss observed on heating up beyond 710° to 1,200 °C, which indicates better thermal stability of the sample in this range.

Fig. 2
figure 2

TG/DT analysis of HAp powder

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X-ray diffraction analysis

The crystalline phase analysis of the HAp powder from fish scale is carried out by XRD studies. The XRD of HAp indicates the visible crystalline nature of typical apatite crystal structures. It is also noticed that crystallographic behavior of HAp resembles to that of XRD pattern of biological apatite [20] which is shown in Fig. 3. However, the pattern of the fish scale-derived HAp powder peaks corresponds to the JCPDS card No. 72-1243 of pure HAp. The highest intensity peak of synthesized powder is in the plane of 211 and it resembles to the pure HAp crystal XRD characteristics. The two minor β-TCP peaks are observed at 31° and 34° after sintering at 1,200 °C.

Fig. 3
figure 3

XRD analysis of HAp powder

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Fourier transform infrared spectroscopy analysis

FTIR spectroscopy is employed to characterize the different functional groups of HAp [Ca10 (PO4)6(OH)2]. FTIR spectrum of HAp powder calcined at 700 °C is recorded in the range of 4,000–400 cm−1 as shown in Fig. 4. The characteristics of FTIR peaks in the wave range 570–632 cm−1 resemble asymmetric bending vibration of P–O band of HAp materials, whereas the peak at 960–965 cm−1 is due to symmetric stretching vibration of P–O band of PO4 3− ion. The spectral data at the wave number values of 876, 1,412 and 1,451 cm−1 suggest the presence of carbonate ion [21] in the prepared HAp materials calcined at 700 °C. The strong peaks at 1,053 and 1,095 cm−1 are ascribed to asymmetric stretching mode of vibration of P–O bands of PO4 tetrahedra [22, 23]. The characteristic frequencies derived from PO4 3− modes are better resolved with increasing temperature. The existence of O–H and C–O bands disappears beyond calcinations temperature of the powder (1,200 °C). But the structural O–H peaks at 1,655 and 3,370 cm−1 are predominant with increasing temperature, whereas the existence of C–O bands is reduced significantly. Therefore, sintered HAp ceramics synthesized from fish scale beyond 1,200 °C is established.

Fig. 4
figure 4

FTIR analysis of HAp powder

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FE-SEM and EDX analyses

The FE-SEM micrograph of fish scale-derived HAp dried powder and fillers scaffolds is depicted in Fig. 5. The morphology of calcined wet ball-milled dried HAp powder shows soft agglomerated ultrafine particles. The biocompatible powder consists of a uniform porous structure upon compacting at 200–250 MPa with porogen (starch granules Merck). The compacted HAp-sintering surface shows distinct grain boundary with uniform porous structure. EDX analysis of the synthesized HAp powder shows that the ratio of Ca/P is 1.71 as depicted in Fig. 6.

Fig. 5
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SEM images of porous HAp scaffold and HAp particles synthesized from fish scale

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Fig. 6
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EDX analysis of HAp particles

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Chemical analysis of Ca/P ratio in fish scale HAp

To perform the chemical analysis, a weighed quantity of hydroxyapatite powder is dissolved in dilute (1:1) nitric acid (HNO3). Calcium content is determined from an aliquot part of the solution by reverse complexometric titration, i.e., by titration of the excess of complexone III (disodium salt of ethylenediamine tetra-acetic acid) with the use of a solution of nickel(II) chloride in ammonia. Murexide (ammonium purpurate) is used as the indicator for titration.

Phosphorus was determined colorimetrically as the phosphovanadomolybdate complex with a UV–VIS Shimadzu Model spectrophotometer using a wavelength of 420 nm. Phosphorus content is determined by the photometric method on the basis of the formation of yellow phosphomolybdic acid and its subsequent reduction to a blue complex compound in a hydrochloric acid solution of thiocarbamide in the presence of copper (II) sulfate. The error of the analysis is 0.5 % Ca and 0.1 % P [24]. The experiment is repeated thrice to ascertain the Ca/P ratio. The ratio of Ca/P is in the range of 1.70–1.73, whereas the EDX experiment shows 1.71 as depicted in Fig. 6.

TEM analysis

Actual particle shape analysis is a complex process in powder technology and till now there is no shape factor available which will clearly differentiate all possible kinds of shape [25]. In spite of these complexity till date, TEM and SEM both instrumentation are highly efficient to determine particle shape and size. In this study, HAp particle size, shape and morphology are observed by TEM images. According to TEM images, small rice-shaped particles are observed in plenty and some spherical large particles are also observed. The fringe pattern of HAp interatomic space is about 0.35 nm as shown in Fig. 7.

Fig. 7
figure 7

TEM images of HAp particles and atomic distribution in fringe pattern

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Mechanical characterization

The compacted samples are optically polished to measure the micro hardness with 100 g load. The average Vickers hardness (HV) of HAp sample is observed about 115 HV. The average porosity is observed about 30–35 %. Compressive stress of the hydroxyapatite samples is tested by UTM (Make: Tinius Olsen, U.K). Sample’s L/D ratios are between 4 and 6. Loads are applied with extension and compression rate of 0.2 mm/s. The ultimate compressive stress of the developed HAp scaffold has reached up to ~0.8 GPa which is better than cortical human bone [26]. Different human skeletal parts and their comparative features of mechanical characterization are shown in Table 1.

Table 1 Mechanical properties of different bone tissues and related materials with references
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Biocompatibility study

In vitro cytotoxicity study of the developed HAp is carried out by culturing RAW macrophage-like cells in a contact mode. RAW cells are cultured in RPMI media and seeded on the six well culture plates at their exponential phase of growth at a density of 105 cells/200 μl. The cells are allowed to attach to the films surface for 6 h in 5 % CO2 incubator at 37 °C. HAp powder samples as well as fillers/plates are utilized for in vitro testing. The samples of different weights (100, 200, 400 μg) are placed into RPMI culture media. RPMI supplemented with 10 % fetal calf serum (FCS) is added to each well to keep the cell-containing samples submerged. The culture plates are incubated for 24 h at 37 °C in a humidified atmosphere of 5 % CO2 in air. Two set of experiments are performed for this cell viability assay. One set of experimental plates is further incubated for more than 48 h in the same atmospheric condition with equal-interval fresh media incorporation in the culture plates. After 72 h incubation in cell culture media samples are stained with trypan blue and observed under inverted microscope as depicted in Fig. 8. In Fig. 8a red trigonal symbol shows living healthy RAW cell lines attached on culture vessels where as yellow arrow indicates the hydroxyapatite particles synthesized from fish scales. Software analysis of images 8(b) distinctly defines the attached cells (blue arrow) and the hydroxyapatite particles (indigo trigonal) as shown in Fig. 8b. Attached living healthy cells prominently show their protoplasm in Fig. 8b. The experimental result reveals the non-immunogenic effect of hydroxyapatite particles toward cell lines (Table 2).

Fig. 8
figure 8

Cytotoxicity evaluation in RAW cell line media and attachment of cells on HAp surface

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Table 2 Results of MTT assay: percentage of cell viable
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On the other hand, second set of experimental plate is utilized for MTT assay. After 24 h incubation, MTT (10 μl) is added to each well at a strength of 10 % (v/v) and incubated for further 4 h at 37 °C. Subsequently, the media containing MTT is removed, and 100 μl of DMSO (dimethylsulfoxide) is added to dissolve the formazan crystals. The plates are agitated for 5 min and read at 550 nm on a scanning multi-well spectrophotometer plate reader (Biorad, USA). t test is performed for statistical significance analysis and a P value of <0.05 is determined to represent a significant difference. The percentage of cell viable is expressed as

$$\frac{\text{Absorbance of treated cells}}{\text{Absorbance of controlled cells}}\;{ \times }\; 100\;\% .$$

Cell attachment study

HAp blocks are used for in vitro cell attachment studies. HAp blocks are sterilized and then RAW cell lines are used in RPMI cell culture media for this study. After 72 h incubation samples cell culture media is decanted and washed thoroughly with phosphate buffer solution (PBS). After washing 2 % paraformaldehyde solution is utilized for cellular fixation. Figure 8c shows SEM image attached cells on to the HAp surface. This study reveals that cells can be attached on the HAp surface which would be utilized as scaffold material for tissue engineering application. Software analysis of Fig. 8c shows the exact attached location of cell lines on hydroxyapatite surface as shown in Fig. 8d. This experimental result shows the bioaffinity of synthesized hydroxyapatite particles toward cell line.

Histological analysis

In the present pilot study, histological analysis [27, 28] is performed in Wistar rats. Wistar rats are easy to handle and their femur is brittle but not fragile during drilling. Skeletally mature 3-months-old female Wistar rats of body weight 150–200 g from the animal house are used for the experiment. The animals are maintained at standard environmental conditions (temperature 22–25 °C, humidity 40–70 % with 12:12 dark/light photoperiod) approved by the Committee for the Purpose of Control and Supervision of Experiment on Animals (CPCSEA) whereas all the experimental protocols are approved by IAEC, CCMB.

Animals were anesthetized with ketamine and xylazine with a dose of 40 mg and 5 mg/kg body weight, respectively. Lateral and median aspect of the femur is cleaned. An incision is made to cut open the skin on the dorsal aspect above the femur. A careful incision is then made to cut open the biceps femoris muscles to expose femur bone. A Pediatric bone driller is used to make a hole of 5 mm in the dorsal surface of femur without touching the bone marrow thus preventing any hemorrhage. The drilled femur of control animal is left unfilled, whereas in experimental group the HAp rods are fixed to fill up the gaps. The muscle layer is stitched with simple interrupted suture using absorbable materials and skin is closed using non-absorbable suture materials. Povidone, iodine ointment is applied externally for 3 days. At the end of the experiment after 3 months, the animals are sacrificed by overdose of CO2. Detail necropsy is done and femur bone is collected and fixed in 10 % formalin [29]. Fixed bone is decalcified with EDTA before embedding in paraffin wax. Fixed and paraffin embedded bones are cut at 5 μm thickness, stained with Hematoxylin and Eosin following standard procedure and examined under light microscope as shown in Fig. 9a.

Fig. 9
figure 9

Histological analysis of femur bone of Wistar rat with HAp prosthetic fillers. a and b shows Bone formation on hydroxyapatite implant (arrow) shows lining cells. Healing is on progress in 3 months sample. Some integration of cells are seen, cell infiltration on materials are also observed (star). Pre-osteoblast cells appear which could be identified by mono nuclear cells wih prominent nulceoli and deeply stained cytoplasm (rhombus). Each bar (thick line) represents 50 μm

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The response of tissues and the implanted material is assessed. Trauma regions are being recovered by healing new cells as cell infiltration on materials is observed. Deeply stained region may conclude the presence of pre-osteoblast cells because this type of cells consists of mono nucleus with prominent nucleoli. In some regions, new cell linings are appeared which may be the result of osteoconduction as shown in blue arrow in Fig. 9b.

Conclusion

  • Labeo rohita scale-derived HAp is almost similar to the structure of pure HAp.

  • The porous HAp scaffold with ~30 % porosity shows considerable improvement in compressive strength ~0.8 GPa which may be suitable for nutrient and biological fluid transportation for in vivo system.

  • Cytotoxicity analysis of HAp particles and MTT assay indicates no cytotoxic effects on macrophage-like RAW cell line media.

  • The histological analysis concludes cell infiltration and integration in HAp fillers which is a good result of bioactivity.

  • Cellular attachment on HAp surface reveals that hydroxyapatite microsphere is able to support cell lines to adhere and proliferate.