ORIGINAL ARTICLE Bali Anatomy Journal (BAJ) 2019, Volume 2, Number 2: 51-57 E-ISSN : 2620-3154 ; P-ISSN : 2621-2021 Potential syringetin-based nHAC/PLGA as bionano bone graft to improved osteoinductivity and osteoconductivity

I Kadek Wahyu Putra Dyatmika*, Jonathan Hubert Prasetya, I Wayan Windi Artha DOI: 10.36675/baj.v2i2.30

ABSTRACT

Background: According to the data of Riskesdas in 2013, fracture which has explicit inclusion and exclusion criteria. incident rate in Sulawesi gets a 16% increase. The most common Discussion: Syringetin-based nHAC usage has some targets like fracture incident happens to elderly age group, and its predominant BMSC. Syringetin-based nHAC affects in increasing the expression cause is a traumatic incident like falls and traffic accident. Treatment of BMP-2 and miRNA-21. BMP-2 and miRNA-21 are factors that take now such as ORIF and External Fixation is far from cheap and has part in osteoblast proliferation and differentiation. That expression a high risk to the patient. Syringetin-based nHAC/PLGA usage can increase is marked by ALP activity and osteocalcin increase. be a new bionano bone graft because it can target specific cells to Conclusion: Significantly, the usage of syringetin-based nHAC/ increase osteoinductivity in bones. PLGA can increase the proliferation and differentiation rate of BMSC Method: This literature review is established by the literature to osteoblast. review method of collecting sources from valid scientific literature

Keywords: Syringetin, nHAC/PLGA, Osteogenesis, Osteoinductivity

Undergradute Program in INTRODUCTION age range of 85 and above in both genders.5 Other Medicine, Medical Faculty, than vertebral fracture (24%), leg fracture (10,5% is Universitas Udayana Fractures are defined as discontinuity of bone the second most common fracture and the third structure and are generally a common occurrence most common is rib fracture (10.3%).5 in the elderly age group; the incidence of fractures 1,2 The cause of the fracture in the report in the elderly tends to increase with age. The from Amin et al. divided into four, namely prevalence of fractures is 4.4% of the population severe trauma (33,4%), moderate trauma of South Sulawesi and 5.8% of in Indonesian 2 (60,2%), pathological (2,1%), and those not population. Increased prevalence of fractures in included in that category (4.3%).5 From this study, South Sulawesi was also reported in Riskesdas severe trauma usually causes leg fractures (20%); Indonesia 2013 by comparing Riskesdas in 2007 moderate trauma and pathological conditions often and 2013; there was a 16% increase in the incidence 2 cause thoracic vertebral fractures (32% and 38% of fractures from 3.7% to 4.3%. The incidence of respectively).5 fractures in South Sulawesi often occurs in the age 2 Treatment of fracture patients can be classified as group 65-74 years (11.1%). In South Sulawesi, the non-operative and operative handlers. Handling of incidence of fractures in Indonesia is lower in the 3 non-operative fractures can be done conservatively young group than the older one. such as elevation, the application using ice, and Gender also influences on fracture rates, studies splints.6 Management of orthopaedic operations of hip fracture incidents in Japan and Singapore *Correspondence to: such as open reduction and internal fixation (ORIF) indicate that the incidence of hip fracture is 2.6 I Kadek Wahyu Putra and external fixation. Treatment after operating and to 3.5 times more common in women.4 There Dyatmika ; Undergradute non-operating fractures are currently only focusing have also been reported increases in Japan over Program in Medicine, Medical on the reduction of short-term complications and Faculty, Universitas Udayana; the past 30 years. The increment was concluded long-term. Both postoperative treatment and non- [email protected] due to the increasing population of the elderly in 4 operation strategies aim to relieve both the pain Japan. Increasing the elderly population occur and venous thromboembolic state.7 However, there in Indonesia, and the prevention and treatment is still no bone graft treatment that has the target of of fractures need to be improved to maintain and increasing osteoinductivity in the fractured bone. improve the quality of life of the elderly in the future. Received: : 16 October 2019 Inducing the osteoblast will improve the process Accepted: 25 Nopember 2019 Data taken from Olmsted County reports the most of forming osteoblast cells. Osteoblast cells will Published: 1 December 2019 frequenting fractures are vertebral fractures with an

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build new bone tissue so that it will reshape the because of its biocompatibility, bioactivity, bone tissue structure to become new. and osteoconductive properties.10 Various Various limitations of the treatment of fractures studies have shown that it can be absorbed offer opportunities for the development of new by osteoblasts and affect the fusion therapies. One of the natural ingredients that have the of osteoclast mononuclear precursors. These effect of increasing osteoinductivity is syringetin. properties make nHAC potentially able to deliver Syringetin is a flavonoid derivative that has drugs directly to the desired tissue and are useful a mechanism of action on improving BMP- as a tool to improve the treatment of several bone 2 and miRNA-21 to accelerate the process of diseases such as osteomyelitis and cancer.11 differentiation and maturation of osteoblast. One of the essential ingredients The low bioavailability of syringetin was of hydroxyapatite derived from natural materials enhanced by the use of encapsulation of HAC/ is an eggshell. Hydroxyapatite (EHA) of eggshell PLGA nanoparticles. Those nHAC / PLGA also has formulation showed excellent biocompatibility and excellent properties as a bone graft for fractures. material properties in vitro. It is a promising graft With these advantages, syringetin-based nHAC/ material with unique features for grafting. It is PLGA has the potential to be a bio-nano bone graft hydrophilic, absorbs fluid and blood around it, in the treatment of fractures. making it easy to handle and put on the surgical site. It can be implemented as a graft material for METHOD grafting bone defect secondary to periodontal disease, trauma, tooth extraction, intra- The study was a literature review that consisted bone defects, sinus removal procedures, and so on.12 of relevant journals from search engines such Poly lactic-co-glycolic acid (PLGA) is a as Pubmed, Google Scholar, and Proquest. copolymer compound which is synthesized Writers searched the keywords “Syringetin”, through the opening of the copolymerization ring “Nanohidroxyapatite”, “PLGA”, “Bone remodelling”, of two different monomer compounds that form “Fracture”, “Osteogenesis”, and “Osteoinductivity”. cyclic dimers (1,4-dioxide-2,5-diones) of lactic acid The inclusion criteria were all syringetin, and glycolic acid. The ratio of the amount between nanohydroxyapatite, PLGA, osteogenesis, lactic acid and glycocalyric is 50:50. PLGA has osteoinductivity. The collected references then excellent biocompatibility and biodegradability noted and analyzed for validity and reliability, properties. It is useful in making nanoparticles.13,14 interpreted and compiled into a scientific literature In reconstructive or regenerative bone review. surgery, ideal material functions not only as a carrier for drug delivery but also as a porous DISCUSSION scaffold for cellular activity. So that the combination Syringetin: Flavonoids Induces Osteogenesis of nHAC with type one collagen and PLGA will be Flavonoids are compounds that can be a useful scaffold for bone cell growth, and drug 15 extracted from various types of fruits and delivery such as syringetin. vegetables. Some flavonoids have been shown to affect osteoblastic diffraction,Fracture healing mechanism and Bone such as daidzein, genistein, , Remodeling , naringin, , and diosmetin. Fracture Healing divided into several steps. First, Syringetin (3,5,7,4’- tetrahydroxy-3’,5’ there is a hematoma and inflammation at the site dimethoxyflavone) is a derivative of flavonoids, of the fracture; second, chondrogenesis begins and which can be found in and khamar. The callus formation occurs (cells that become vascular concentration of syringetin found in grapes reached bone was also at this stage); third, osteoblast cells 3.22%.8 begin to form hard bones and connect the two Hsu et al. research showed that syringetin could broken bone segments (osteogenesis); fourth, increase BMP-2 synthesis and activate SMAD1/ bone remodelling occurs with the ended result of a 16-18 5/8 and ERK1/ 2. These effects can contribute to its stronger lamellar bone structure. The four steps activity to induce maturation and differentiation above can also be divided into two phases, namely of osteoblasts followed by an increase in bone mass.9 the anabolic phase (the cell recruitment phase and matrix mineralization) and the catabolic phase Nanohydroxyapatite and Its Application (the accumulation phase of mineral accumulation 18 Nanohydroxyapatite collagen (nHAC) and vascularisation). is a nanoparticle delivering drugs for Bone remodelling belongs to the catabolic phase. bone therapy. It is frequently applied In that phase, osteoclasts begin to absorb the

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matrix which has been mineralized during with the insertion of the nHA (scaffold) into the the osteogenesis phase.18 In this phase suspension modality with a vacuum suction of too, osteoblasts and osteoclasts work side 0.1 MPa for 10 minutes, and mixed with a vortex by side (coupled remodelling); structure mixer for 10 minutes.19 Syringetin in mixed and vascularisation marrow hematopoietic tissue suspension which is not bound to nHA can be remodelling extensive also occur in this phase separated after centrifugation of the suspension. (Figure 1).18 Many syringetins that bind to nHA can A critical compound in the fracture healing and be remodelling by subtracting the initial bone remodelling is Bone Morphogenic Protein total syringetin with the rest of the syringetin. 2 (BMP-2). Mouse with the lack of expression of As discussed in the introduction, BMP-2 reported having a defect in differentiation no bone grafting treatment increases and the proliferation of osteoblasts.18 Lack the osteoinductivity of the fractured bone. of BMP-2 expression has also been reported Administration of the syringetin modality will to cause osteoblasts not activated at fracture be carried out in conjunction with bone grafts healing sites.19 Based on the above data, it can be inserted during ORIF (open reduction and internal concluded that lack of BMP-2 expression causes fixation) orthopaedic surgery. Bone graft with disruption of osteoblast production and function, this syringetin into the category of bone graft and as a result, interferes with the process of biosynthetic. osteogenesis (callus not fully formed) and bone remodelling is inhibited. Mechanism of Action of Syringetin-Based nHAC/PLGA Nanohydroxiapatite Construction and Syringetin Effect on Osteoinductivity Administration Mechanism Syringetin has osteoinductivity property in Nanohydroxyapatite (nHA) will be used as a inducing differentiation of pre-osteoblast into scaffold for the syringetin modality. Some reasons late-osteoblast. An induction increase in pre- for selecting nHA have been discussed earlier in this osteoblast differentiation will increase new bone article. There is also research by Wang et al. states tissue formation. Osteoinductivity property of that nHA can distribute evenly and in a uniform syringetin comes from the induction of BMP-2 and form.19 In the first 24 hours, modalities constructed Runx2 expression. Neither the specific target nor with nHA, as much as 55% and the remainder were the mechanism of BMP-2 induction by syringetin released slowly over a period of three days (the is known until now. Ya-Ling Shu et al. stated that modality used by Wang et al. Was insulin); it states administration of syringetin to human fetal the ability of nHA in the equitable distribution of osteoblastic (hFOB) cell and MC3T3-E1 increase modalities, and the ability of sustained release is their BMP-2 expression.9 good considering fracture healing usually lasts long Basic helix-loops-helix (bHLH) protein enough.19 and E-box protein binding will activate BMP-2 Construction begins by preparing expression. Both bHLH protein and E-box protein the syringetin pattern in suspension, then proceed specifically regulates BMP-2 gene expression on mesenchymal stem cell. Expressed BMP-2 then will bind to BMP-2 receptor and form heterodimer complex. Heterodimer complex activates two types of signal transduction that are Smad-independent and Smad-dependent. In Smad-independent signal transduction, there is p38 activation by mitogen- activated protein kinase (MAPK) that enables Runx2 to control mesenchymal stem cell differentiation and TAK-1 (TGF-β Activated Kinase-1) to regulate bone formation.10 In Smad-independent signal transduction, TGF-β inducted receptor binds TRAF6 and ubiquitin to the cytoplasm to activate TAK-1 (TGF- β Activated Kinase-1) and MAPK (Mitogen- Activated Protein Kinase). TAK-1 and MAPK activation play a role in regulating mesenchymal stem cell differentiation and bone formation. Figure 1. Bone remodeling Activated TAK-1 and MAPK trigger JNK and p38

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phosphorylation. p38 and JNK will translocate into nucleus plasma and there they will form a complex with these transcription factors (Ras-Raf-MEK-Erk, MAPK JNK-c-Jun N terminal kinase, activating transcription factor-2 (ATF-2), p38 MAPK, SH2 domain-containing sequence A (SHCA), and extracellular signal-regulated kinase (Erk)); these complexes will then increase gene expression.10 Aside from Smad-independent activation path, heterotetramer complex can activate Smad path through R-Smad phosphorylation (Smad 1,5, and 8). Smad 1,5, and 8 can induce other TGF-β transducing protein signals. R-Smad activation leads to heteromeric complexes forming with C-Smad and their translocation into the nucleus. Those heteromeric complexes will bind with some other transcription factors [Runx2/Cbfa1 (core- binding factor alpha 1), Osx (Osterix), Dlx5, and Msx2 (msh homeobox homolog 2)]. Lastly, those molecules play a role in osteogenesis induction (Figure 2). The increment of BMP-2 expression marks syringetin effect. Besides, there are increase in alkaline phosphate (ALP) activity, an increase in osteocalcin expression, and type-1 collagen synthesis.10, 21 Ya-Ling Shu et al. research showed that there is an increase of BMP-2 expression on MC3T3-E1 (osteoblast precursor cell line from Mus musculus) mice and hFOB cell (osteoblast cell line from human fetus cell). Increase in syringetin regulation can be observed 3 hours after injection of 20µM, and its maximal expression is noted after 12 hours. After 12 hours, the rise of BMP-2 expression follows a time- dependent and dose-dependent manner. Syringetin inducts BMP-2 dependent pathway (Figure 3).9 Increase in osteoblast proliferation can be tested by observing ALP and osteocalcin activity. ALP is a biological marker secreted in the early phase of Figure 2. Mechanism of bone morphogenetic protein 2 (BMP-2)10 osteoblast proliferation. Osteocalcin is the other biological marker that plays a role in evaluating osteoblast proliferation in the termination phase. When syringetin is injected into MC3T3-E1 mice and hFOB cell, ALP and osteocalcin increase is expected. ALP activity increases after 20µM syringetin administration with the dose- dependent manner in 48 hours and 72 hours. ALP increase marked the stimulation of pre-osteoblast proliferation into early osteoblast. After that, early osteoblast will develop into late osteoblast and then osteoblast itself (Figure 4).9 The development phase of the late osteoblast is marked biologically by osteocalcin. Syringetin administration on MC3T3-E1 mice and hFOB cell can increase late osteoblast to osteoblast cell Figure 3. Level of BMP-2 after 12 hours treatment with various concentration proliferation rate. This increase is marked by the 21 (1, 5, 10, 20µM) at MC3T3-E1 and hFOB. rise in osteocalcin secretion after 48 hours and

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research compared syringetin administration with noggin administration as a negative control that will be tested with immunoblotting analysis. The result showed that SMAD1/5/8 and Erk1/2 was much thicker on syringetin administration than that of noggin (BMPR-2 inhibitor) (Figure 5).23

Nanohydroxyapatite effect on increased osteoinductivity An important characteristic that nanohydroxyapatite have is osteoconductivity. Osteoconductivity helps in new bone tissue formation on a surface in an ongoing fracture Figure 4. Effect syringetin increase ALP activity after 12 hours of treatment healing. Nanohydroxyapatite has hydrophilic 21 with various concentration nature and helps osteogenic cell be more adhesive, proliferative, and differentiative on forming bone tissue. Osteogenic cells are much more prone to apoptosis in hydrophobic surface than that of a hydrophilic. Tong et al. study used human osteoblast-like cell (SaOS-2) that was cultured into carbonated nHA (CHA) on polyhydroxybutyrate-co-hydroxy valerate (PHBV).24 Proliferation, morphology, and ALP were evaluated in 14 days. After 14 days, the result showed that ALP was found significantly increased in CHA/PHBV than in PHBV. Although both CHA/PHBV and PHBV shows increased Figure 5. Activation of Smad 1/5/8 and ERK1/2 after treated with syringetin adhesiveness, proliferation, and cell distribution. in MC3T3-E1 and hFOB cell analysed using immunoblotting This reflects the use of CHA/PHBV increase ALP assay.21 expression in cells.24 Zhang et al. study are about nHA/PCL activity with weight ratio difference on nHA and PCL to human fetal osteoblast (hFOB).25 72 hours of dose-dependent manner syringetin The result showed that there was ALP activity and administration. This shows syringetin can work mineral matrix synthesis increase compared to 25 on early phase to the late stage of proliferation and control. differentiation Figure( 4).9 According to Arumugam et al. research, PLGA Role in Half Time Enhancement syringetin administration on mMSCs in osteoblast nHAC/PLGA use for encapsulation could differentiation process can Runx2 expression. increase the half time of syringetin. Xing Wang Runx2 is a bone transcription factor that also plays study used a compound which was the same a role in osteoblast differentiation transcription characteristic as syringetin and showed the use of marker much like ALP and osteocalcin. Syringetin nHAC/PLGA increase its half time. nHAC method administration is proven to increase miR-21 for encapsulation showed that there was a 93% expression, which in turn will inhibit Smad7 compound release in 24 hours. While in nHAC/ expression. Smad7 inhibition leads to increased PLGA showed 46% compound release in 24 hours 21, 23 Runx2 expression due to Smad7 naturally inhibit and constant release for 5 days (Figure 6). Runx2.23 Increased expression of BMP-2 on MC3T3-E1 Citocompatibility dan bioactivities of nHAC/ and hFOB cell by 20µM dosage of syringetin PLGA administration can increase phosphorylation of The effect of nHAC/PLGA can affect the SMAD1/5/8 and Erk1/2 activation. Syringetin differentiation process of BMSC. Arumugam et al. therapy will increase phosphorylated SMAD1/5/8 research the differentiation and maturation degree after 3 hours, and its maximal effect is attained in 12 of MSC using ALP and osteocalcin (OCN) as hours. There is also an increase of Erk1/2 activation biological markers. The result shows an increase in after 3 hours of syringetin administration. That ALP on day three until day fourteen after nHAC/ PLGA administration; there is no difference in

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marked by increased activity of ALP and an increase in OCN expression. Increased expression of miRNA-21 will inhibit the expression of Smad7 which plays a role in inhibiting the activation of Runx2. Its usage as encapsulation one may increase the half-life of syringetin and increase the rate of proliferation and differentiation of cells stem cells marked by an increase in ALP and OCN. The existence of an increase in the rate of proliferation and the length of half-life can be a treatment for fractures in the elderly because in the elderly, there is a decrease in the rate of bone remodelling. So expect nHAC/PLGA-based syringetin can be innovation bionano bone graft to pen n Subscribe fractures in the elderly.

DISCLOSURES There is no conflict of interest and no grants stated 23 Figure 6. nHAC/PLGA effect on substances release. by the authors.

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