1. Introduction
Bone is a highly vascularized, dynamic and diverse tissue, which forms the skeletal framework of the body. It is essential for support, protection of delicate vital organs, locomotion, mineral homeostasis and possess load-bearing role [1]. The loss or deterioration of bone alongside with large or critical-sized bone defects cause premature disability and reduction in the quality of life. Though bone grafts [2] augment critical-sized bone defects and promote bone regeneration, it suffers a lack of integration and fails to meet the rising demand for the grafts. This paves the way for the need of engineered bone tissue and thus bones tissue engineering (BTE) which aims in providing the repair process by using the synergistic combination of biomaterials, cells and factor therapy [3]. In BTE, the scaffolding materials with the following ideal properties of osteogenicity, osteoconductivity, biocompatibility, and biodegradability are employed [4].
Flavanoids, due to their anti-oxidant property are exploited in cancer therapy, and this property can also enhance osteoblastogenesis and inhibits osteoclastogenesis [5]. This property of the flavanoids [6] can be synergistically combined with the scaffolds to promote bone regeneration. Silibinin is a plant derived, polyphenols flavinoligan seen in silymarin, derived from the seeds and fruits of the milk thistle plant, Silybum marianum [7]. The wide range of biological activities possessed by Silibinin is anti-hepatotoxic [8], anti-inflammatory [9], inhibition of oxidative stress [10] and anti-cancer properties [11]. Apart from these anti-hepatotoxic and anticancer effects, recent studies have claimed the effect of Silibinin on osteogenic differentiation of human bone marrow stem cells [6]. Studies on the effects of Silibinin on the osteoblast formation enhancement [5, 12] and osteoclast formation inhibition [13] have been the root to study the effects of Silibinin on osteogenesis. Silibinin, due to its bulky multi-ring structure and poor oral bioavailability, is hydrophobic in nature. The low aqueous solubility of Silibinin limits its bio-availability and water solubility, thus limiting its clinical role [14]. This leads to the need of an approach, to overcome drug-associated problems by increasing the bioavailability of hydrophobic drugs and improving the efficacy.
The delivery of hydrophobic compounds can be done in an improved manner by the use of Nano-drug delivery systems such as nanoparticles, nanosuspension, nanomicelles and nanoemulsion [15, 16]. To avoid drug associated difficulties, natural polymers are opted for the preparation of nanoparticles due to its biodegradable nature [17]. Thus, chitosan (CS), a promising mucoadhesive and permeation enhancer can be used to encapsulate the drugs by

using TPP (Sodium-Tri poly phosphate) a suitable cross-linker [18]. CS is a natural, semi-crystalline, cationic polysaccharide, composed of repeating units of d-glucosamine and N-acetylglucosamine [19, 20]. It is biocompatible and is attributed to the structural similarity of ^lycosaminoglyeans (GAGs). It also possesses antibacterial [21] and biodegradable properties [22]. To facilitate a sustained delivery of drugs, devoid of initial burst release from :he nanoparticle, the drug-loaded nanoparticle can be encapsulated in scaffolds [23]. These scaffolds preferably made up of natural polymers due to their non-toxic, non-immunogenic ind well-interconnected structure [24]. The two natural polymers, Alginate (Alg) and Gelatin [Gel), possess chemical similarity with the bone extracellular matrix (ECM) and hence, can DC suggestive for fabrication, as a blending of polymers increases its performance in an effective way [25, 26].
Alg is an anionic polymer, hydrophilic, biocompatible, and biodegradable at normal ?H. It is a linear copolymer with homo- polymeric block of (l-4)-linked p-d-mannurate (M) ind its C-5 primer a-1-guluronate (G) residue) [27, 28]. Sodium alginate can form scaffolds within a relatively short period and can easily be manipulated to regulate the level of porosity 29]. Gel is biodegradable, bioresorbable, cytocompatible, and non-antigenic protein. The martial hydrolysis of collagen yields Gel. Gel mimics the ECM, due to the presence of the iU3D sequences. It is widely used as a delivery matrix, in wound dressing and as scaffolds 29, 30]. The degradation properties are strongly dependent on the scaffolding materials, and ailoring of the scaffold degradation is obtained through adjusting materials properties [31].
Biomaterial and phytochemicals could mediate several intracellular signals which in urn could activate several gene regulators of osteogenesis such as microRNAs (miRNAs) 32-37]. miRNAs are a novel class of posttranscriptional gene regulators which are 19-25 lucleotides in length [38]. miRNAs can regulate bone remodelling by acting as either legative or positive regulator of osteoblast differentiation by targeting their genes. Several )hytochemicals have been shown to regulate the expression of miRNAs in various cells. The expression of miRNAs is finely orchestrated, being up regulated or down regulated to ■egulate the differentiation stage of each bone cell, resulting in a characteristic miRNA signature in osteogenesis [39-41]. Thus, this work was aimed in tailoring the sustained and )rolonged release of Silibinin from the CS encapsulated and Alg/Geln incorporated scaffolds, ind in determining its effect on osteoblast differentiation at the cellular and molecular levels n vitro.

2. Results
The SCN (Silibinin entrapped in chitosan nanoparticles)' were synthesized by ionic gelation technique where 0.5 % (w/v) of chitosan (CS) was dissolved in 1% (v/v) acetic acid and was crosslinker with 0.2% (w/v) of TPP solution in the ration 4:1, with three different concentrations of Silibinin (20 uM, 50 uM, and 100 uM) dissolved in DMF. The scaffolds incorporated with SCN were prepared by freeze drying method with the use of 5% (w/v) Alg (Alginate) dissolved in distilled water and 5% (w/v) Gel (Gelatin) dissolved in distilled water at 60° C mixed in equal proportion, with the addition of the SCN and sonicated to obtain a homogenous dispersion. The scaffolds were crosslinked in 100 mM CaCb overnight at 4° C and with 1% glutaraldehyde solution for 30 min and washed with distilled water to remove the excess glutaraldehyde followed by lyophilization at -40°C for 48 hrs. 2.1 SEM, EDS, Zeta potential and Silibinin entrapment release analyses of SCN and Alg/Gel-SCN Scaffolds
The SEM analysis was done to determine the particle size of the CN and SCN and to
study the external morphology and porous structure of the Alg/Gel, Alg/Gel-CN, and
Alg/Gel-SCN scaffolds. The results showed CN and SCN of size 215 ± 16 nm and 259 ± 20
nm (Fig. 1), respectively and they were found to be spherical in shape and when the
concentration of Silibinin increased in SCN, their size was increased (Table 1). Porosity of
the scaffolds determines the cell infiltration [42] and cell permeability [43]. The SEM
analysis of the scaffolds (Fig. 2A) revealed a highly porous and interconnected architecture
with the pores uniformly spread, and they were in the range of 120-150 fim. The EDS gives
an insight on the purity and.confirmation of the sample elements. The elemental compositions
of the scaffolds were identified as Carbon (C), Oxygen (O), Sodium (Na), Calcium (Ca),
Chloride (CI) by EDS analysis (Fig. 2B) contributed by the atoms of chitosan, Silibinin,
Alginate, Gelatin, glutaraldehyde and the crosslinker CaCl2.

Using zeta potential measurements, the surface charge and thereby the stability of the prepared nanoparticle systems was determined. The zeta potential values of+42.5 mV, +44.8 mV, +46.3 mV and +48.3 mV were obtained for the CN, 20 uM SCN, 50 uM SCN and 100 fiM SCN, respectively (Fig. 3) (Table 1). The positive zeta potential of CN are attributed to the presence of free, unconjugated amine groups on the surface of CN, and the higher value indicates the good stability of nanoparticles [44]. The zeta values of the SCN were similar to CN indicating that the addition of Silibinin did not affect the charge or the stability of the nanoparticles. The efficiency of the CN entrapped with Silibinin was measured as the % of entrapment efficiency (EE). It was calculated indirectly by determining the Silibinin content in the supernatant of the SCN that was stored during the preparation of the SCN. The % of EE of CS was found to be 94,89 ± 2.72 at 20 uM Silibinin, 92.22 ± 0.34 with 50 uM Silibinin, and 97.66 ± 1.13 with 100 uM Silibinin (Table 1).
12 XRD and FT-IR analyses of SCN and Alg/Gel-SCN Scaffolds
The X-ray diffractogram of pure CS showed the semi-crystalline nature of it, and the X-ray diffractogram of Silibinin showed the sharp and intense peaks at 14.3°, 16.1°, 17.4°, 19.5°, 20.5° and 24.5° which indicate the crystalline nature of Silibinin (Fig. 4A). On the formation of the CN, there was the complete disappearance of the diffraction peaks and the intense characteristic peaks of Silibinin disappeared in the diffractogram of the SCN confirming the amorphization of CS, followed by the amorphization of Silibinin resulting in the loss of structural arrangement of the lattice during its entrapment into CN. When the drug is hydrophobic, this phenomenon is especially preferred in drug targeting [10]. The ranges of less intense peaks in Alg/Gel are contributed by the semi-crystalline Gel, and Alg is amorphous in nature. The x-ray diffractogram of Alg/Gel-CN and the Alg/Gel-SCN were similar to that of Alg/Gel, suggesting that the semi-crystalline nature of the scaffolds were not affected by the addition of the amorphous CN and SCN, respectively.'To investigate the chemical interaction between S, CS, Alg/Gel and to identify the functional groups, FTIR analysis was carried out (Fig. 4B). The FTIR spectrum of pure, standard Silibinin showed the characteristic peaks at 3453.9 cm"1 (-OH stretching), 2478 cm"1 (C-H stretching), 1634.8 cm"1 ;CO stretching), 1509 - 1469 cm"1 (aromatic C=C stretching), and 1270.3 cm"1 (C-O-C stretching). At the expense of the free hydroxyl bands, the broad intense band for H-bonded ghepolip,OI^stretching^wasgeer^.|n^tl[ie regioJn/2g0Q-36Qpf^rn"lJ In the. Ghifos'an spectra, the

strong and wide peak in the 3500-3300 area was attributed to hydrogen-bonded O-H stretching vibration. The peaks of N-H stretching from the primary amine and type II amide were overlapped in that region. The peak for an "asymmetric stretch of C-O-C was found at 1150 cm"1, and the peak at 1317 cm"1 showed the C-N stretching vibration of type I amine. The FT-IR spectrum of pure sodium tripolyphosphate showed the characteristic bands at 1218 cm-1 (P-0 stretching), 1156 cm-1 (symmetrical and asymmetric stretching vibration of the PO2 groups), 1094 cm-1 (symmetric and asymmetric stretching vibration of the PO3 groups) and 892 cm-1 (P-O-P asymmetric stretching). The FT-IR of CN confirmed tripolyphosphoric groups of TPP linked with ammonium groups of CS in the nanoparticles. In CN, the tip of the peak of 3438 cm"1 had a shift to 3320 cm-1 indicating an augmentation of hydrogen bonding. Also, the peaks for N-H bending vibration of amine I at 1600 cm-1 and the amide II carbonyl stretch at 1650 cm"1 shifted to 1540 cm"1 and 1630 cm-1, respectively. The crosslinked CS showed a P=0 peak at 1170 cm-1. These results have been recognized as the linkage between phosphoric and ammonium ion [45]. In the FT-IR of SCN, the characteristic peaks of Silibinin and CN were observed. The characteristic peaks of Alg at 3373 cm"1 (O-H stretching) and 1036 cm"1 (C-O-C stretching) were observed. The characteristic peak of Gel at 1553 cm"1 was due to the overlapped peak caused by C-N and C=N stretching contributed by glutaraldehyde crosslinking that takes place by the reaction of available free amine groups on Gel with glutaraldehyde [46]. The FT-IR spectra of Alg/Gel-CN and Alg/Gel-SCN had the characteristic peaks of S, CN and Alg/Gel (Fig. 4B). 2.3 Swelling, biodegradaion, protein adsorption and bio-mineralization of Alg/Gel-SCN scaffolds
The swelling ability of the scaffolds is a critical parameter as it relates to the water retention ability of the scaffolds. This relates to the absorption of the body fluids, water and facilitates the transport of nutrients [46] and deep cellular infiltration [47]. It was seen that the swelling ability of the scaffolds increased initially and then decreased after 24 h, when studied in PBS at 37° C (Fig. 5A) leading to the increase in pore dimensions and more cell attachment. Biodegradation of the scaffolds on implantation is essential as this aid in the formation of the new bone [4]. The rate of degradation of the scaffolds in PBS containing lysozyme at different time periods showed that the addition of CN or SCN to the Alg/Gel scaffolds significantly increased the rate of degradation. Silibinin has no significant role in determining the degradation (Fig. 5B) and the gradual increase in the degradation of scaffolds over the time which attributed to the role of the cross-linkers employed in the preparation of ^ the^scaffoJds that facilitate, a-controlled degri^atign^ w^hrch-jis^an- imgortanrpaijarneter in BTE.

Adsorption of proteins on the scaffolds helps in the cellular interaction, thus influencing the cell adhesion, cell spreading and the late cellular events, such as proliferation and differentiation [48]. Natural polymers aid in enhanced protein adsorption [22, 49]. Thus Alg/Gel-CN and Alg/Gel-SCN were found to possess increased protein adsorption than the control group without the CN (Fig. 5C). The fate of cell-adhesion onto the scaffolds and the growth followed by cascade of biological properties are dependent on the amount of protein adsorbed [34] and hence, the synthesized scaffolds with increased protein adsorption are viable for BTE.
Bio-mineralization is the ability to form new bone by the deposition of mineral salts on the surface when implanted and exposed to body fluid [50]. The exogenous biomineralization study was carried out in vitro by incubating the scaffolds in SBF [51] for 7 d. The deposition of the apatite layer was examined and studied by SEM, EDS and XRD analyses (Fig. 6). The SEM analysis revealed that the scaffolds (Alg/Gel) have the potential to undergo biomineralization. There was increased deposition onto the Alg/Gel-SCN scaffolds which can be resulted due to increasing in surface area by SCNs (Fig. 6A), and the EDS analysis further showed elemental peaks for calcium along with the peaks for the scaffold composition (Fig. 6B). The XRD patterns of the biomineralized scaffolds revealed characteristic diffraction peaks of hydroxyapatite (JCPDS-09-0432). The peaks were found to be intensified in the Alg/Gel-SCN corresponding to the degree of crystallinity and thus enhanced biomineralization (Fig. 6C). This can be attributed by the release of Silibinin in the medium, aiding in increased deposition of apatite. 2.4 Silibinin release from Alg/Gel-SCN Scaffolds
The release of Silibinin from Alg/Gel-SCNs was determined for a time period of up to 15 d (Fig. 7). Silibinin was found to be released devoid of initial burst effect, which could be due to the incorporation of SCNs in the Alg/Gel scaffolds. The percentage of Silibinin released from the Alg/Gel-SCN was about 60% on the 15 day. Further, the samples obtained on the 15th day were subjected to HPLC analysis, and the result confirmed the presence of Silibinin qualitatively. Similar results to the standard Silibinin were obtained, with two peaks at the retention times 21.89 min and 22.31 min corresponding to the isoforms of Silibinin (Silibinin A and Silibinin B), respectively. It has been reported that the initial burst effect for drug release was due to the drug present on the surface for diffusion [52]. The release of Silibinin from a nanocarrier was also found to be sustained and pH-dependent, and the maximum percentage release of Silibinin from the nanocarrier was seen when exposed to pH . 7.-4-"1 .PBSJ53}.. From-Qurr-resultSi we suggest $iat the; s^eaffojd act%d:as3awarder for the

release of Silibinin from the SCN of scaffolds, thus preventing the initial burst release, followed by the sustained release from degradation of the scaffolds.
2.5 Biological characterization of Alg/Gel-SCN scaffolds
MTT assay was done to check if the synthesised scaffolds are toxic to the mouse mesenchymal stem cells (C3H10T1/2). Cells were directly seeded on the scaffolds and subjected to MTT assay as described in Methods section. The Alg/Gel, Alg/Gel/CN and Alg/Gel-SCN scaffolds were found to be non-toxic to C3H10T1/2 cells (Fig. 8).
2.6 Cellular level differentiation ofmMSCS to osteoblasts
To determine the effect of the release of Silibinin on MSCs differentiation towards osteoblasts at the cellular level, C3H10T1/2 cells were allowed to grow onto the scaffolds for 7 d. The ALP activity was determined by the BCIP-NBT assay, where the ALP active cells stained blue-violet in colour. There was increased in blue-violet staining of cells in the Alg/Gel-SCN scaffolds compared to the Alg/Gel-CN scaffolds (Fig. 9A). The quantification of ALP activity at 620 nm showed a significant increase in the Alg/Gel-SCN scaffolds compared to the Alg/Gel-CN scaffolds (Fig. 9B). To confirm the differentiation of the MSCs to osteoblasts, a direct staining of the mineralized deposits on the scaffolds was carried out, as the ALP activity is not only limited to osteoblasts. The calcium deposits on the scaffolds are an indication of progressive differentiation of MSCs into osteoblasts and in vitro bone-formation, and they are stained using alizarin red stain. The result showed that there was increased alizarin stained calcium deposits with Alg/Gel-SCN scaffolds compared to control (Alg/Gel-CN) (Fig. 10A). The qualitative analysis by determining the absorbance at 405 nm showed a significant increase in mineralization where cells were seeded onto Alg/Gel-SCN compared to control (Fig. 10B). The silibinin released from Alg/Gel-50uM SCN showed enhanced mineralization on comparing with Alg/Gel-20 \\M SCN and Alg/Gel-100 uM SCN. Thus, the significance of Silibinin on the promotion of MSC differentiation into osteoblasts at the cellular level was determined, and the results also identified the osteoinductive nature of the Alg/Gel-SCN scaffolds.
2.7 Molecular level differentiation ofmMSCS to osteoblasts
The role of Silibinin released from the Alg/Gel-SCN in the differentiation of the mMSC to osteoblasts at the molecular level was. determined by the expression of the osteoblast differentiation marker genes. Runx2 is a bone transcription factor required for determination of the osteoblast lineage, and its expression is upregulated in osteoblasts. Runx2, during the early stages of osteoblast differentiation, triggers the expression of maior~.bpne associated

matrix genes [54]. ALP, an early marker gene in differentiation osteoblasts, directs the cells towards matrix formation before mineralization [55]. The late markers of differentiated osteoblasts are OC and COL-1. The process of mineralization is controlled by OC, the non-collagenous protein of the bone, and it characterizes the mature cells of osteoblastic lineage by binding with the hydroxyapatite crystals [56, 57]. COL-1 on the other hand is the most abundant protein in the organic bone matrix (about 90%) [58].
The mRNA expression levels of ALP, type I collagen (COL-1), osteocalcin (OC) and Runx2 genes was determined after treating the cells with scaffolds for 14 d (Fig. 11). The Runx2 mRNA level was significantly increased when cells were treated with Alg/Gel-20 uM SCN and Alg/Gel-50 uM SCN compared to Alg/Gel-CN (Fig. 11 A). The mRNA expression of ALP (Fig. 1 IB) and OC (Fig. 1 ID) genes was found to be increased with Alg/Gel-50 uM SCN and Alg/Gel-100 pM SCN compared to control. The mRNA expression of COL-1 (Fig. 11C) was found to be increased with Alg/Gel-50 uM SCN compared to control. In addition to finding the expression level of Runx2 at mRNA level, we determined its protein expression level by western blot analysis. Similar to the mRNA expression of Runx2, the protein expression level was also found to be increased in the Alg/Gel-SCN compared to Alg/Gel-CN (Fig. 12A). The blots were scanned and quantified after normalization with a-tubulin for the relative expression of Runx2 protein (Fig. 12B), and the result was consistent with its mRNA expression (Fig. 6A). Among the prepared groups of scaffolds, it is seen that Alg/Gel-50 uM SCN upregulated the mRNA levels of the osteoblast differentiation marker genes, which indicated the osteo-conductive nature of the prepared scaffolds. Hence, these results suggest that role of Silibinin in osteogenesis both at the early stage and maturation stage. 2.8 Effect of Silibinin on pre-miRNA(s) expression
Recent studies indicated the role of miRNAs controlling gene expression under physiological and pathological conditions [38, 39]. miRNAs are short non-coding RNAs (20-22 nucleotides) that act as post-transcriptional regulators of gene expression and play an essential role in the regulation of osteogenesis by controlling the expression of several signalling components, transcription factors and cofactors [59-62]. To identify the miRNAs which are regulated during osteoblast differentiation by Silibinin, we selected 5 miRNAs (miR-20b, miR-30c-l, miR-93, miR-221, and miR-410) which have been shown to target various genes that participate in osteoblast differentiation [63-70]. To determine the expression pattern of pre-miRNAs (precursor miRNAs) in C3H10T1/2 cells by the scaffolds containing Silibinin (Alg/Gel-50 uM SCN), real-time RT-PCR analysis was carried out. ,_. &ince .the. rAlg/GeI-50^ uM- *SCN« scaffolds y uppegulate^j ^the expressi§n4 of osteoblast

differentiation markers (Figs. 12, 13), we used these scaffolds. The expression of pre-miR-20b and pre-miR-410 was found to be significantly increased when cells were treated with AIg/Gel-50 uM SCN compared to Alg/Gel-CN scaffolds for 14 d; whereas the expression of pre-miR-30c-l and pre-miR-221 was found to be significantly decreased (Fig. 13). Pre-miR-20b was shown to enhance osteogenesis by co-repressing of PPARy, BAMBI, and CRIM1 and activating BMPs/Runx2 signalling pathway [63]. Thus, the increased expression of pre-miR-20b could result in enhanced osteogenesis. Similarly, the increased expression of pre-miR-410 indicated its positive role by targeting VEGF and sequentially maintaining osteogenesis [64, 65]. Pre-miR-93 has been studied to suppress osteogenic differentiation by targeting SMAD5 [66], but we found no change in its expression by Silibinin treatment. Pre-miR-30c-l was known to target Runx2 [67, 6$], and Runx2 expression was found to be upregulated by Silibinin (Fig. 13). It has been shown that downregulation of pre-miR-221 triggers osteogenic differentiation [69, 70]. In the current study, the expression of pre-miR-221 was significantly downregulated by Silibinin. All the miRNAs except pre-miR-93 reported in this study suggest that the release of Silibinin from Alg/Gei-50 uM SCN enhanced osteogenesis by either up or down regulation of miRNAs resulting in the activation of BMP/SMAD/RUNX2 signalling pathways, which are known to promote osteoblast differentiation [39-41, 59]. The regulation of miRNAs by phytochemicals and biomaterials can be helpful in understanding the underlying molecular mechanisms and its functional aspects of a cell-material interaction cascade [32-37]. The primers used in the study are summarized in Table 2.

3. Summary
The addition of the SCN to the Alg/Gel scaffolds enhanced the physiochemical properties and influenced the material characterization studies. These scaffolds were found to be biocompatible as they were non-toxic to the mMSCs. The release of Silibinin from the scaffolds especially Alg/Gel- 50 uM SCN stimulated differentiation of mMSCs towards osteoblasts at the cellular and molecular levels. Further, the release of Silibinin from Alg/Gel-50 uM SCN regulated the expression of miRNAs that controlling the BMP pathway and thus, promoted osteoblast differentiation. Hence, the sustained and prolonged release of Silibinin from the fabricated scaffolds possesses the osteo-conductive and osteo-inductive properties, which strongly suggest its candidature for bone tissue engineering. TENT OFF I C E CH£ U H AI .ZQ / <3 5 / 2 G 1 7 1. 1 ": 3 4

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Claims:
1. A nano-drug delivery system consisting of a flavonoid entrapped in cationic polysaccharide nanoparticles and incorporated into a biocomposite scaffold containing an anionic polymer and a polymer obtained by the partial hydrolysis c collagen was prepared by freeze drying technique for bone tissue engineering.
2. The flavonoid according to claim 1, wherein a plant derived flavonoglycan is der from the seeds of the milk thistle plant is Silibinin.
3. According to claim 2, the concentrations of Silibinin used are 20 uM, 50 uM and uM in DMSO.
4. The nano-drug delivery system according to claim 1, the cationic polysaccharide chitosan crosslinked with sodium tripolyphosphate.
5. According to claim 4, chitosan of 0.5% (w/v) and sodium tripolyphosphate of 0.2 are used in the ratio 4:1 for the formation of the nanoparticles.
6. According to claim 1, wherein the scaffold material of anionic polymer is alginati and the partial hydrolysis of collagen is gelatin.
7. The scaffolds according to claim 6, wherein the concentration of alginate is 5% (i and the concentration of gelatin is 5% (w/v).
8. The scaffolds according to claim 7, wherein alginate is crosslinked with 100 mM CaCb and gelatin is crosslinked with 0.5% (v/v) glutaraldehyde.
9. The process according to claim 1 tailors the release of silibinin from the chitosan nanoparticles encapsulated in the bio-composite scaffold containing alginate and gelatin.
10. The sustained release of Silibinin according to claim 9, wherein promoted bone formation evidenced at cellular and molecular levels, and stimulated the BMP pathway that involves in bone formation by regulation of the expression of microRNAs.