Enhanced Intrafibrillar Mineralization of Collagen Fibrils Induced by

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Biological and Medical Applications of Materials and Interfaces

Enhanced Intrafibrillar Mineralization of Collagen Fibrils Induced by Brush-Like Polymers Le Yu, Ian J Martin, Rajeswari Kasi, and Mei Wei ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b10234 • Publication Date (Web): 06 Aug 2018 Downloaded from http://pubs.acs.org on August 11, 2018

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ACS Applied Materials & Interfaces

Enhanced Intrafibrillar Mineralization of Collagen Fibrils Induced by Brush-Like Polymers Le Yu†,#, Ian J. Martin‡,#, Rajeswari M. Kasi‡,∫,§,*, Mei Wei†,§,* †

Department of Materials Science and Engineering, ‡Department of Chemistry, ∫Polymer

Program, §Institute of Materials Science, University of Connecticut, Storrs, CT 06269, USA

# L Yu and I J Martin contributed equally to this work. *

Authors to whom correspondence should be addressed.

Prof. M Wei: [email protected]; Tel: +1 (860)-486-5003 Prof. R M Kasi: [email protected]; Tel: +1 (860)-486-4713

Keywords: biomimetic mineralization, collagen, intrafibrillar and extrafibrillar, brush polymer, polyethylene glycol, polyacrylic acid, mineralization mechanism

ACS Paragon Plus Environment

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Abstract Biomimetic mineralization of collagen fibrils is an essential process since the mineralized collagen fibers constitute the basic building block of natural bone. To overcome the limited availability and high cost of the non-collagenous proteins (NCPs) that regulate the mineralization process of collagen, commercially available analogs were developed to replicate sequestration and templating functions of NCPs. The use of branched polymers in intrafibrillar mineralization applications has never been explored. In this work, two novel carboxyl-rich brush-like polymers, a carboxylated polyethylene glycol terpolymer (PEG-COOH) and a polyethylene glycol/ polyacrylic acid copolymer (PEG-PAA), were synthesized and modified to mimic the sequestration function of NCPs to induce intrafibrillar mineralization of collagen fibrils. It was found that these synthetic brush-like polymers are able to induce intrafibrillar mineralization by stabilizing the amorphous calcium phosphate (ACP) nanoprecursors and subsequently facilitating the infiltration of ACP into the gap zone of collagen microfibrils. Moreover, the weight ratios of mineral to collagen in the mineralized collagen fibrils in the presence of these brush-like polymers were 2.17 ± 0.07 for PEG-COOH and 2.23 ± 0.03 for PEG-PAA, while it as only 1.81 ± 0.21 for linear PAA. Plausible mineralization mechanisms using brush-like polymers are proposed, which offer significant insight on the understanding of collagen mineralization induced by synthetic NCPs analogs.


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1. Introduction Natural bone is a composite of crystalline nano-hydroxyapatite (HA, Ca5(PO4)3(OH)) reinforced within collagen fibrous matrix, and its basic building blocks are mineralized collagen fibers. It is structurally described from nanometer to millimeter scales as a seven-hierarchical-level organization with mineralized collagen fibrils as its second level of hierarchy.1-2 Therefore, biomimetic mineralization of collagen fibril is a critical process in preparing bone grafting materials, as it directly contributes to the superior mechanical and excellent biological properties of the material.3-5 Moreover, it is reported that the mineralized collagen scaffolds possess better osteoconductive properties and exhibit higher levels of osteogenic gene expression than collagen alone scaffolds.6 Mineralized collagen fibers are more effective as sustained delivery vehicles of drugs or growth factors like Chlorhexidine and BMP-2.7-8 Thus, it is imperative to better understand the mineralization mechanisms to further adjust the mineral content in the collagen matrix. In the biomineralization process, HA crystals first form within the gap zone between abutting collagen molecules, and then spread through the collagen fibrils, leading to arrays of HA nanocrystals embedded within and oriented along the fibrils with a 67 nm periodic pattern, known as intrafibrillar mineralization.1, 9-11 Subsequently, HA crystals grow and attach onto the surface of collagen fibrils, resulting in extrafibrillar mineralization.10 It is widely accepted that the biomineralization of collagen is well regulated by a series of non-collagenous proteins (NCPs). However, NCPs only compose about 3 wt% of the organic components.11-13 It is hard to take advantage of natural NCPs to reproduce the decent hierarchy in bone due to the limited availability and its high cost.11,

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Recently, researchers have discovered that regulation of

calcium phosphate microstructure during biomineralization is impacted by two major factors: (1) sequestration of the calcium and phosphate ions into amorphous calcium phosphate (ACP)


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nanoprecursors and (2) templating of HA nucleation, growth and guided orientation.15-17 An alternative strategy has therefore been developed in order to explore synthetic analogs to replicate the sequestrating and templating functions of NCPs in the biomineralization process. Polyanionic polymers, such as polyaspartic acid (PASP) and polyacrylic acid (PAA), are employed as the sequestration analogs of NCPs to suppress bulk crystallization and to stabilize the amorphous phase of calcium phosphate and therefore attain intrafibrillar mineralization.10, 18 Previous studies have shown that the carboxyl groups of the sequestration analogs play an essential role in stabilizing ACP by readily attracting calcium ions.19-20 These sequestration analogs are usually used in conjunction with phosphate-based templating analogs such as sodium tripolyphosphate (TPP), which possesses high affinity for divalent ions, to obtain highly ordered intrafibrillar mineralization.21-22 However, these analogs are not all available for clinical applications at the present and the mineralization degree of collagen using PASP and PAA as analogs is diminished due to their linear structure and limited availability of carboxyl groups.23 It is desired to develop new synthetic biomaterials that can induce biomimetic mineralization of collagen fibrils and are capable of adjusting mineral content to serve different purposes. Brush polymers are composed of side-chain polymers that are densely grafted onto a polymer backbone and they have garnered much attention due to their outstanding control over chemical functionality, architecture, and size.24 Brush polymers have a variety of applications in organic electronic devices,25 biosensors,26 and low friction surfaces.27 Additionally, bottlebrushlike systems have been utilized as effective boundary lubricants to reduce the frictional force between the sliding surfaces of mammalian synovial joints.28 The architecture of brush polymers varies with grafting density, side chain and backbone flexibility, and monomer repulsion within a copolymer. The steric repulsion between the grafted side chains increases backbone rigidity and


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lowers entanglements between polymer chains. Brush polymers are synthesized by a “graft-to”,29 “graft-from”,30 or “graft-through”31 approach. The “graft-to” method entails attaching polymer chains to a substrate; however, steric hindrance provided by attachment of initial polymer chains results in overall low grafting density. “Graft-from” consists of an initiator-functionalized surface where highly grafted polymer chains are grown, but results in a higher dispersity of chain lengths due to a monomer concentration gradient that occurs as the polymerization proceeds.32-33 The “graft-through” method solves these issues by end-capping a macromonomer (MM) with a polymerizable unit capable of highly efficient polymerization. This enables complete grafting of side-chains to every backbone monomer unit while maintaining a low dispersity of chain lengths. Brush-like copolymers (BLCPs) prepared by the “graft-through” technique exhibit greater flexibility than brush polymers due to their lower grafting density.34 “Graft-through” by ring-opening metathesis polymerization (ROMP) using norbornene-derived (NB) MMs for BLCP preparation allows for the unique tailoring of each MM by size and functionality prior to polymerization.35-36 Preparing NB MMs by controlled-radical polymerization techniques such as reversible addition-fragmentation chain transfer (RAFT) permit fine tuning of side chain lengths with diverse chemical compositions.26,

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This allows for precise control over polymer

architecture and chemical composition by independently varying the backbone and side chain length. Additional post-polymerization modifications of BLCPs allow for greater diversity of chemical composition and polymer properties. Poly(amidoamine) (PAMAM) dendrimers are also extensively studied and commercially used in the biomineralization field due to its potential in mimicking natural NCPs.2,

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Although there has been a wealth of studies of PAMAM dendrimers in comparison to novel brush-like systems, the synthetic modularity of the brush-like polymers allows for greater control


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over architecture and degree of functionality. The synthesis and purification of higher generation dendrimers are challenging to prepare due to increased steric hindrance at the periphery. Conversely, as mentioned above, brush-like polymers may exhibit much greater variation in their side-chain length and/or backbone length, depending on the specific application and do not require extensive purification.41 The advantages of brush-like systems over linear PAA are most likely due to an increased backbone rigidity caused by steric hindrance of the grafted side-chains. This results in lower chain entanglements, which in turn provides an opportunity for an increase in mineral content of the mineralized collagen fibrils. Taking great advantages of brush-like polymers over commercially available products like PAA and PAMAM, a principal goal of the current study is to synthesize and further modify two different brush-like polymers, a carboxylated polyethylene glycol terpolymer P(NBMPEG-rNBPEG-r-NBPEG-COOH) (TP-1; PEG-COOH) and polyethylene glycol/ polyacrylic acid copolymer P(NBMPEG-r-NBPAA) (CP-1; PEG-PAA), to induce intrafibrillar mineralization of type I collagen fibrils by mimicking the role of sequestration functions of NCPs. The mineralization performances induced by these brush-like polymers were compared to those of linear PAA, which is currently one of the most popular synthetic sequestration analogs. The results shed a new light on enhancing the mineral content as well as explaining the intrafibrillar mineralization mechanism of collagen fibrils.

2. Materials and Experiments 2.1 Materials for mineralization Type I collagen was extracted from rat-tails, purified based on the protocol reported by Rajan et al. and further modified by our group.42-44 Sodium chloride (NaCl; > 99%), potassium phosphate


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dibasic trihydrate (K2HPO4·3H2O; > 99%), and sodium tripolyphosphate (TPP, 85%) were purchased from Acros Organics. Sodium bicarbonate (NaHCO3; > 99%) and polyacrylic acid (PAA, Mw = 2,000 Da) were purchased from Sigma-Aldrich. Calcium chloride anhydrous (CaCl2; > 99%), magnesium chloride hexahydrate (MgCl2·6H2O; > 99%), sodium hydroxide (NaOH; 1N) and 4-(2-hydroxyethyl)-1-piperazinneethanesulfonic acid (HEPES; > 99%) were purchased from Fisher Scientific.

2.2 Synthesis of Monomers, Copolymer, and Terpolymer The synthetic routes to afford TP-1 and CP-1 can be found in Schemes S1 and S2, respectively. Monomers norbornene-poly(ethylene glycol) (NBPEG),24 norbornene-poly(ethylene glycol) methyl ether (NBMPEG),24 norbornene-poly(tert-butyl acrylate) (NB-p(t-BA)), and macroRAFT agent norbornene trithiocarbonate (NB-TTC)25 were synthesized from modified procedures. The detailed synthetic procedures and characterization of monomers can be found in sections 2.1, 2.2 and 2.6 in the Supporting Information (SI).

1

H NMR spectra and peak assignments of the

monomers NBMPEG, NBPEG, NB-p(t-BA), and a mass spectrum of NBMPEG are presented in Figures S1, S3, S6, and S2, respectively. Ring-opening metathesis polymerization (ROMP) of NBPEG and NBMPEG was performed to synthesize P-1, the precursor polymer of TP-1. ROMP of NBMPEG and NB-p(tBA) was also used to synthesize P-2, the precursor polymer of CP-1. The corresponding chemical structures of TP-1 and CP-1 are listed in Table 1. Both polymerizations used a modified second generation Grubbs catalyst in CH2Cl2. Post-polymerization modifications of P-1 and P-2 were carried out by a maleic anhydride ring opening reaction and hydrolysis of tert-butyl groups to afford TP-1 and CP-1, respectively. The detailed synthetic and modification procedures


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of P-1, P-2, TP-1, and CP-1 can be found in sections 2.3-2.5, and 2.7-2.8 in the SI. 1H NMR spectra and peak assignments of P-1, TP-1, P-2, and CP-1 are presented in Figures S4-5 and S78, respectively.

2.3 Self-assembly of intrafibrillar mineralized collagen fibrils Based on our previously established protocol, intrafibrillar and extrafibrillar mineralized collagen fibrils were fabricated in the presence of different analogs.14,

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Briefly, different

sequestration analogs (PAA or brush polymers) were added to a modified simulated body fluid (m-SBF) to form stabilized amorphous calcium phosphate (ACP) nanoprecursors. The m-SBF was prepared as reported previously: NaCl, K2HPO4·3H2O, MgCl2·6H2O, and CaCl2 were added in chilled sterile deionized water (DIW) in the order as they are listed, and the ion concentrations were adjusted according to Table S1.46-47 Concentrations of PAA and/or brush polymers in each m-SBF were adjusted according to Table 2. To investigate the capability and efficacy of intrafibrillar mineralization induced by brush-like polymers with lower concentration, PAA/PEG-COOH and PAA/PEG-PAA groups were designed. To maintain comparable weight percent and ionic strength, half PAA was also added in the other two groups. Type I collagen stock solution (4 mg/mL) was diluted to 0.2 mg/mL using PAA- and/or brush polymercontaining m-SBF. TPP (1.2 wt%), the templating analog, was simultaneously added to the mixture to template the hierarchical arrangement of mineral within collagen fibrils. Then the pH of each solution was titrated to 7.2 by addition of HEPES (4-(2-hydroxyapatiteethyl)-1piperazineethanesulfonic acid), NaHCO3 and NaOH. The preparation of mixtures was conducted on ice, where the resulting solutions experienced a two-temperature process by leaving them at 25 °C for 1 h, and then transferring them to a water bath at 37 °C for another 23 h. Such prepared


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fibrils were spun down using a centrifuger (Legend XTR, Thermal Scientific, USA) at 5000 rpm for 10 min, and the resulting precipitates were further freeze-dried for 7 days in a lyophilizer (Free Zone, Labconco, USA) to remove the residual moisture within the fibrils.

2.4 Materials characterization 2.4.1 Monomer and polymer characterization 1

H NMR spectroscopy was performed on a Bruker DMX 500 MHz NMR spectrometer with

CDCl3 or DMSO-d6 as solvent and the peak of CDCl3 at 7.24 ppm or the peak of DMSO-d6 at 2.50 ppm is used as an internal standard. Gel permeation chromatography (GPC) was performed using a Waters 1515 coupled with a PL-ELS1000 evaporative light scattering (ELS) detector and a Waters 2487 dual wavelength absorbance UV-Vis detector using dimethylacetamide or tetrahydrofuran as eluent and polystyrene (PS) standards for constructing a conventional calibration curve. Mass spectrum of NBMPEG is obtained with Micromass Quattro-II triple quadruple mass spectrometer equipped with an electrospray ionization (ESI) positive ion mode source.

2.4.2 Fibril characterization The morphologies and intrafibrillar mineralization formations of all five groups of fibrils that listed in Table 2 were observed using transmission electron microscopy (TEM, JEOL JEM-2010, Japan) at an accelerating voltage of 80 kV. For TEM sample preparation, 100 µL fibrilcontaining solution (experienced two-temperature process) was dropped onto a formvar carbon coated copper grid, rinsed with DIW, and dried at ambient temperature without staining. To check the potential of the brush-like polymers to be employed in preparation of tissue


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engineering scaffolds, the collagen fibrils that mineralized in the presence of PAA or brush-like polymers were centrifuged and then subjected to a freeze-drying process. The morphologies for these fibrils were then examined using TEM (FEI Talos F200X S/TEM, USA) at an accelerating voltage of 200 kV in the UConn/Thermo Fisher Scientific Center for Advanced Microscopy and Materials Analysis (CAMMA). After freeze-drying, the fibrils were re-suspended into DIW and 100 µm solution was dropped onto a TEM grid until dried in air without staining. To investigate the intrafibrillar mineralization mechanism in the presence of brush-like polymers, ACP nanoprecursors formed in the presence of PEG-COOH or PEG-PAA were observed using TEM and HRTEM via FEI Talos F200X S/TEM in the CAMMA. Briefly, after pH titration (before experiencing the two-temperature process), 100 µL mixture was dropped onto a TEM grid, rinsed with DIW and finally dried in air without staining. The elemental mapping distribution of above-mentioned nanoprecursors and fibrils were also obtained from FEI Talos F200X S/TEM, which is equipped with a Super-X silicon drift detector (SDD) system allowing for rapid acquisition of energy-dispersive X-ray spectroscopy (EDS) data. The intrafibrillar spacing distance and fibril diameter were measured from the TEM images using Nano Measure 1.2 and expressed as mean ± standard deviation. Fourier transform infrared spectroscopy (FTIR) was used to determine the functional groups of all the five groups of freeze-dried samples. The spectra were acquired using a Nicolet Magna 560 FTIR spectrometer (Artisan Technology Group, USA) with an attenuated total reflectance (ATR) accessory. The FTIR-ATR spectra were acquired over the range of 4000-500 cm−1 with a resolution of 4 cm−1 and a total of 32 scans. Thermogravimetric analysis (TGA) was performed using a Q500 TGA analyzer (TGAQ500, TA Instrument, New Castle, USA). About 15 mg freeze-dried collagen fibrils from each


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group were placed in a platinum pan and heated from room temperature to 700 °C at a rate of 5 °C min−1 in air to determine the amount of minerals in the collagen fibrils in the presence of different analogs.

2.4.3 Statistical analysis Statistical results were shown as mean ± standard deviation (SD) and statistically significant differences (p) between brush-like polymer groups compared to PAA group were performed by unpaired T-test using GraphPad Prism 5.

3. Results and discussion 3.1 Synthesis and characterization of brush-like polymers. Several norbornene appended macromonomers were synthesized with PEG or poly(t-BA) side chains capable of post-polymerization modifications to achieve carboxyl-rich brush-like polymers. Reversible addition−fragmentation chain transfer (RAFT) polymerization was used to synthesize the poly(t-BA) macromonomer due to its narrow molecular weight distribution and exceptional control over molecular weight, which resulted in a narrow polydispersity of 1.08. ROMP of the norbornene endcapped macromonomers yielded the precursor polymers P-1 and P2 which exhibited characteristic narrow molecular weight distributions with polydispersities of 1.13 and 1.08, respectively. Carboxyl group incorporation in P-1 was performed by ring-opening of maleic anhydride by terminal hydroxyl groups of the PEG side chains to afford TP-1. Complete hydrolysis of the tert-butyl groups of P-2 by trifluoroacetic acid were successful in achieving PAA side chains in CP-1. A summary of the weight percentage, number of side chain repeat units, theoretical Mn, and polymer dispersity for the synthesized PEG-PAA brush


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polymers can be found in Table 3. The extent of carboxylic acid functionalization of TP-1 was determined by comparing the sum of the integrations of the olefinic peaks at 6.19 and 6.36 ppm of the reacted maleic anhydride to the integration of the norbornene olefinic backbone at 5.29 ppm in Figure S5. An example calculation of the weight percentage and number of repeat units of carboxylic acid functionalized side chains can be found in section 2.5 in the SI. The Mn of TP1 was determined by the addition of maleic anhydride to four NBPEG side chains of P-1. The hydrolysis of P-2 to yield CP-1 was performed using 50 equiv. of trifluoroacetic acid to 1 equiv. of tert-butyl group. Complete hydrolysis of tert-butyl groups was shown to occur when comparing the 1H NMR spectra of P-2 and CP-1 in Figure S7 and S8, respectively. The emergence of a broad singlet at ~12.2 ppm (peak a of Figure S8) is indicative of the carboxylic acid protons of CP-1 and the disappearance of the tert-butyl protons at 1.43 ppm (peak a’ of Figure S8 inset) of P-2 corroborate this assumption. The loss of tert-butyl protons to P-2 provides a theoretical Mn of 44.3 kDa for CP-1. After confirming the chemical composition of the brush-like polymers, the dialyzed and freeze-dried TP-1 and CP-1 are ready to be used as sequestration analogs for biomimetic collagen mineralization.

3.2 Intrafibrillar mineralization of collagen fibrils in the presence of brush-like polymers. It is previously reported that needle-shaped carbonated apatite precipitated randomly on the surface of collagen fibrils in the absence of sequestration and templating analogs, and periodic banding patterns were not observed.14-15 Therefore, to achieve intrafibrillar mineralization of collagen fibrils, a dual-analog system with TPP as the templating analog while PAA and/or brush-like polymers (TP-1 and CP-1) as sequestration analogs (Table 2) were used in the current work to replicate the dual functions of NCPs. To investigate the capability of above-synthesized


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brush-like polymers in inducing intrafibrillar mineralization, TEM images from all five groups listed in Table 2 were collected. Figure 1 shows typical TEM morphologies of un-stained collagen fibrils in the presence of different sequestration analogs. Distinct electron-dense periodic banding patterns (D-banding) can be observed within the un-stained collagen fibrils from all groups, indicating that minerals had deposited within the gap zone of collagen fibrils and thereby reproduced the D-banding pattern similar to those normally observed in natural bone. To exclude the possibility of TPP induced intrafibrillar mineralization, a control group without any addition of PAA or brush-polymers was added. As shown in Figure S9, no Dbanding patterns are detectable at the presence of TPP but the absence of PAA or brushpolymers. Therefore, it can be concluded that our brush-like PEG-COOH terpolymer (TP-1) and PEG-PAA copolymer (CP-1) are capable of inducing intrafibrillar mineralization of collagen fibrils in the absence of linear PAA. This implies the capacity of these synthetic brush-like polymers in replicating the sequestration function of NCPs. FTIR spectra were obtained from freeze-dried samples to observe the conformational functional groups of these mineralized fibrils and the results are shown in Figure 2. Specifically, the broad peak around 3400 cm−1 is associated with water absorption in the fibrils. The peaks at 1649 cm−1, 1553 cm−1, and 1188 cm−1 are assigned to major characteristic peaks of amide I, II, and III bands of type I collagen.21, 48 The vibrational modes of PO43− were observed with the doublet at approximately 1089 cm−1 and 1022 cm−1 corresponding to the triplet ν3 antisymmetric P-O stretching mode.49 A weak peak at 964 cm−1 corresponds to the ν1 nondegenerate P-O symmetric stretching mode, and the peaks at 600 cm−1 and 560 cm−1 are assigned to the ν4 O–P–O bending mode.50-51 It is noted that a gradual split of the single peak at 580 cm−1 into two peaks at 600 and 560 cm−1 implying the transformation of ACP to HA.8, 52 Therefore,


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the existence of these two peaks in turn suggests the crystallization of apatite in the mineralized collagen fibrils. In addition, the peaks at 1410 cm−1 and 875 cm−1 are typical vibration bands of CO32+, indicating small amount of carbonate substitution in the apatite lattice, which is similar to the mineral phase in natural bone.48, 53 In summary, the FTIR spectra obtained from all five groups of samples exhibit typical vibrational modes of carbonate apatite and collagen. TGA was performed on the freeze-dried fibrils to investigate the mineralization efficiency of the synthetic brush-like polymers. As shown in Figure 3A, the initial 8-12% weight loss in each sample occurs between 30-150 °C, which is assigned to the evaporation of physically absorbed water on the surface of fibrils. This is also called dehydration.54 The second stage has approximately 27-30% weight loss in each sample occurring between 150-700 °C, which is assigned to the decomposition of collagen molecules within the fibrils. The final residual is about 58-64 wt%, which are the minerals. It is obvious that the overall mineral contents in the mineralized collagen fibrils were significantly enhanced by up to 10% using synthetic brush-like polymers, which were approximately 63.9 ± 0.18 wt% for PEG-PAA and 61.6 ± 0.25 wt% for PEG-COOH as sequestration analog compared to that of 57.6 ± 1.11 wt% for commercial linear PAA. It is also to be noted that natural bone is a biocomposite comprised of approximately 65 wt% minerals, 25 wt% organic materials, and 10 wt% water.55 The mineral content in the brushlike polymers is in good agreement as that in natural bone. Every group has different weight percentages of absorbed water. To exclude the possibility that the difference in final weight percentage is caused by different level of water absorbed, dry-weight of each sample at T=150 °C was normalized to 100%. The replotted TGA curves (Figure 3B), show that the group with linear PAA had the most weight loss while the ones with brush-like polymers had the least loss, confirming that the use of brush-like polymers significantly improved the mineral content in


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collagen fibrils. Figure 3C shows the mass ratio of mineral to collagen within the fibrils that calculated from the dry-weight, suggesting that the mass ratio was enhanced by over 20% when the linear PAA in the dual-analog system was substituted partially or all by the brush-like polymers to induce intrafibrillar mineralization of collagen fibrils (1.81 ± 0.21 for PAA group, while 2.17 ± 0.07 (p = 0.11) and 2.23 ± 0.03 (p = 0.006) for PEG-COOH and PEG-PAA group, respectively). Although it is unable to identify intrafibrillar and extrafibrillar minerals from the TGA analysis, it confirms that the brush-like polymers are capable of inducing mineralization of type I collagen by playing the role as a sequestration analog. Moreover, the substitution of linear PAA with brush-like polymers can significantly increase the mineral content within the mineralized collagen fibrils. Mineralized collagen fibers are frequently used to construct tissue engineering scaffolds, and one of the most common techniques used to fabricate scaffolds is freeze casting. It is important to explore the potential of the brush-like polymers to be used as sequestration analogs to fabricate tissue engineering scaffolds. Therefore, the self-assembled mineralized collagen fibrils were lyophilized. The bright field TEM images along with the high angle annular dark field (HAADF) pictures of freeze-dried fibrils synthesized using PEG-COOH or PEG-PAA brush polymers as a sequestration analog compared to those of using commercial linear PAA are presented in Figure 4. In addition to very small amount of minerals deposited on the surface of collagen fibrils (extrafibrillar mineralization), periodically D-banding patterns with a spacing distance of approximately 67 nm (n > 100) are clearly observed from all samples, indicating that both the extrafibrillar and intrafibrillar minerals have been kept intact after freeze-drying. The inset in Figure 4C shows the selected area electron diffraction (SAED) pattern collected from PEG-PAA induced fibrils suggesting that the minerals deposited within the collagen fibrils are


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either nano-crystalline or amorphous. This corresponds to the result reported by Hu et al,48 indicating no ring pattern can be observed in the intrafibrillar mineralized collagen fibrils though clear ring patterns of polycrystalline hydroxyapatite can be detected from both extrafibrillar mineralized collagen fibrils and intrafibrillar mineralized collagen scaffolds. Moreover, the diameter of the collagen fibrils slightly expanded from 141 ± 12 nm (n=108) for the PAA group to 146 ± 14 nm (n = 78; p = 0.09) and 145 ± 10 nm (n = 98; p = 0.05) for the PEG-COOH and PEG-PAA groups, respectively. The increase of the diameter of the collagen fibrils could be attributed to the fact that more minerals were incorporated into the fibrils,56 which is in agreement with the fact that the brush-like polymers enhanced intrafibrillar mineralization compared to linear PAA. Overall, these results not only suggested that the brush-like polymers have a great potential to be used for fabrication of mineralized collagen fibrils and scaffolds, but also significantly promoted intrafibrillar mineralization of collagen fibrils.

3.3 Intrafibrillar mineralization mechanism induced by brush-like polymers. A polymer induced liquid precursor (PILP) concept has been well accepted to explain the intrafibrillar mineralization mechanism when polyanionic polymers like PAA or PASP were used. Briefly, it was believed that ACP nanoparticles first bind to PAA molecules that stabilize ACP complex (PAA-ACP nanoprecursors) adjacent to the collagen fibrils. The small size and fluidic nature of PAA-ACP nanoprecursors allow them to infiltrate into collagen fibrils to obtain intrafibrillar mineralization.14,

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However, as shown in Table 3, unlike the linear PAA that

possesses a molecular weight (Mn) of only 2 kDa, the theoretical Mn of both brush-like polymers synthesized in the current work are above 40 kDa. This implies that the brush-like polymers are unlikely to infiltrate into the gap zone of the collagen fibers according to the size exclusion


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effect.58 Besides, the branch structure also limits their mobility that further reduces the possibility of these brush-like polymers penetrating into the collagen fibers. Thus, it is necessary to look into the underlying mechanism of intrafibrillar collagen mineralization induced by brush-like polymers. Figure 5A shows the TEM image of the nanoprecursors formed in the presence of PEGPAA. Many small droplets were found to accumulate at the joints of the network formed by the polymerized brush-like polymers. The network formation of the brush-like polymer was confirmed by the TEM images (Figure S10). In neutral environment (DIW at pH 6.4) without any addition of ions, round-like nanoparticles with a diameter of 10 ± 3 nm (n=273) were observed (Figure S10A). The nanoparticle formation may have occurred due to the hydrophobic poly(norbornene) backbone comprising the core and the hydrophilic/ deprotonated PEG-COOH side chains as the outer layer of the nanoparticles in the polar solvent. However, a network structure was observed when the brush polymer was added into a collagen-free m-SBF solution at a pH of 7.2 (Figure S10B), excluding the possibility that the observed network was formed by collagen. In contrast to brush polymer, no network structure can be detected when linear PAA was dissolved in the collagen-free m-SBF solution at pH = 7.2 as shown in Figure S10C. At pH = 6.4 ( pH > 3.2), deprotonation of the COOH groups result in electrostatic repulsion of the grafted side-chains,59 forcing the polymer into spheres as seen in Figure S10A. Introducing the brush-like polymer into the ion-rich m-SBF solution may be neutralizing the COO- moieties which consequently lowers the electrostatic repulsions of the side-chains,60 resulting in the network-like formation seen in Figure S10B. The enlarged TEM and HAADF images of the nanoprecursors (Figure 5B/5C) show that each droplet consists of many small nanoparticles. No obvious crystal lattice can be observed with HRTEM (Figure 5D), suggesting the amorphous


17


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nature of the nanoparticles. The EDS mapping results (Figure 5E-H) confirms that the nanoparticles are composed of calcium phosphate. Similar results were also observed from the TEM images of the nanoprecursors formed in the presence of PEG-COOH, as shown in Figure S11. It can be concluded that amorphous calcium phosphate (ACP) droplets mostly forms in the close vicinity of the joints formed by the network formation of brush-like polymers. As these fluidic droplets are mostly smaller than 40 nm in diameter, they are capable of infiltrating collagen fibrils by capillary action and electrostatic attraction to fill in the gap zones between the collagen molecules and spaces between the microfibrils.8, 61 A schematic illustration of the intrafibrillar mineralization mechanism induced by the brush-like polymers is proposed in Figure 6. The network of the brush-like polymer (either PEG-COOH or PEG-PAA) initially forms within the collagen-containing m-SBF solution. On one hand, at the physical network junctions of the brush like polymers, the availability of carboxyl groups is enhanced, which in turn readily attract calcium ions at these sites. Subsequently, phosphate ions are further recruited by the calcium ions, which form a fluidic calcium phosphate nanocluster. The electrostatic attraction between the carboxyl groups and Ca ions inhibits the nucleation of HA crystals that in turn gives a stabilized amorphous mineral structure, namely ACP nanoprecursors as shown in Figures 5 and S11.62 On the other hand, the self-assembly of collagen simultaneously takes place and TPP, the highly negatively charged templating analog, accumulates in the highly positively charged gap zone of collagen fibrils.9, 13 Since TPP is a much stronger penta-anionic chelating agent to ACP compare to the carboxyl groups of brush-like polymers,63 the brush-like polymer stabilized ACP nanoprecursors infiltrate into the gap zone of collagen microfibrils by both capillary action and electrostatic attraction once they are in the vicinity of collagen microfibrils. Finally, aggregation, solidification, and


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partially crystallization of these infiltrated ACP minerals start in the gap zone under the influence of the amino acid side chains of collagen fibrils that give a clear D-banding pattern, namely intrafibrillar mineralization.9, 64 However, the promotional effect of the brush-like polymers on the intrafibrillar mineralization compared to PAA is still in discussion. Unlike linear PAA stabilized ACP nanoprecursors, one possible reason is that the architecture of the brush-like polymers enables greater accessibility of carboxyl groups, producing more stabilized ACP clusters. Another possibility is that the brush-like polymers are able to form a physical network rendering them unlikely to infiltrate into the gap zone of the collagen fibrils together with ACP, leaving more spaces in the gap zone allowing for more ACP accumulation. Therefore, this provides a feasibility to enhance the final mineral content in the collagen fibrils.

4. Conclusions Two novel carboxyl-rich brush-like polymers, a carboxylated polyethylene glycol terpolymer (TP-1) and a polyethylene glycol/ polyacrylic acid copolymer (CP-1), were synthesized and showed great potential as a platform for the preparation of collagen tissue engineering scaffolds. By replicating the sequestration function of NCPs, these brush-like polymers successfully induced obvious intrafibrillar mineralization of collagen fibrils. More importantly, TGA data showed that the mineral contents in the mineralized collagen fibrils produced in the presence of brush-like polymers were significantly higher compared to that induced by linear PAA. By investigating the formation of ACP nanoprecursors, reasonable mineralization mechanisms in the presence of brush-like polymers were also proposed.

Supporting Information


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Supporting information includes details of materials used, experimental procedures, characterization for the synthesized brush-like polymers, compositions of m-SBF, and TEM data of the collagen fibrils and ACP nanoprecursors.

Acknowledgements LY and MW acknowledge NSF grants (CBET-1133883 and CBET-1347130). IJM acknowledges fellowship from Department of Education GAANN Program (P200A150330). RMK and IJM thank Dennis Ndaya for useful conversations regarding the synthesis of the brushlike polymer systems.

Disclosures Dr. Wei has an ownership interest in OrteoPoniX LLC, which is developing and commercializing biomaterials for orthopedic applications.

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Table 1. Brush-like random copolymers P(NBMPEG-r-NBPEG-r-NBPEG-COOH) (TP-1) and P(NBMPEG-r-NBPAA) (CP-1) with their corresponding chemical structures. Polymer

TP-1

P(NBMPEG-r-NBPEG-r-NBPEG-COOH)

Chemical structure x

Entry

O

O O

O

O

O 21

OH

m

O

O O

O

OH

O

n

21

O O

O

O

O 17

CP-1

P(NBMPEG-r-NBPAA)

n

ph

n

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Table 2. Sample labels and corresponding concentrations of different sequestration analogs. Sample name PAA PAA/PEG-COOH PEG-COOH PAA/PEG-PAA PEG-PAA

PAA (mg/mL) 0.8 0.4 ---0.4 ----

P(NBMPEG-r-NBPEG-rNBPEG-COOH) (mg/mL) ---0.4 0.8 -------


P(NBMPEG-r-NBPAA) (mg/mL) ---------0.4 0.8

24

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Table 3. Polymer composition and molecular weight. Entry

Polymer

Weight Percentage a, # of Repeat units b

Mn, theo c kDa/mol;

NBMPEG

NBPEG

NBPEG-

NBPAA

ÐM(PDI) d

--

42.01

COOH TP-1

P(NBMPEG-r-NBPEG-

67.05, 28

21.35, 8

11.60, 4

(1.13)

r-NBPEG-COOH) CP-1

a

P(NBMPEG-r-NBPAA)

Weight percentage and

b

54.58, 20

--

--

45.42,

44.29

20

(1.08)

# of repeat units for TP-1 are determined by 1H NMR integrations of the peaks at 3.35,

3.60, and 6.19-6.36 ppm corresponding to NBMPEG, NBPEG, and NBPEG-COOH respectively. c Theoretical Mn calculated by addition of maleic anhydride to P-1 and complete hydrolysis of tert-butyl groups in P-2 for TP-1 and CP-1, respectively. d ÐM(PDI) of TP-1 and CP-1 prior to post-polymerization modification.


25


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Figure captions

Figure 1. TEM morphologies of intrafibrillar mineralized collagen fibrils in the presence of different sequestration analogs: PAA (A1, A2); PAA/PEG-COOH (B1, B2); PEG-COOH (C1, C2); PAA/PEG-PAA (D1, D2); and PEG-PAA (E1, E2).

Figure 2. FTIR spectra of intrafibrillar mineralized collagen fibril in the presence of different sequestration analogs.

Figure 3. (A) TGA curves (B) Replotted TGA curves using dry-weight (150 °C) as reference; (C) mass ratio of mineral and collagen of intrafibrillar mineralized collagen fibrils in the presence of different sequestration analogs.

Figure 4. Bright field (A-C) and HAADF TEM (D-F) images of freeze-dried fibrils using PAA (A/B) or PEG-COOH (C/D), and PEG-PAA (E/F) as sequestration analog. The inset in C suggests that the minerals deposited are either nanocrystalline or amorphous.

Figure 5. TEM (A-C), HRTEM (D) images and EDS mapping (E-H) of ACP nano-precursor formed in the presence of PEG-PAA copolymer (CP-1).

Figure 6. Schematic illustration of the intrafibrillar mineralization mechanism of collagen fibrils induced by brush-like polymers.


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Figure 1. TEM morphologies of intrafibrillar mineralized collagen fibrils in the presence of different sequestration analogs: PAA (A1, A2); PAA/PEG-COOH (B1, B2); PEG-COOH (C1, C2); PAA/PEG-PAA (D1, D2); and PEG-PAA (E1, E2). 127x52mm (300 x 300 DPI)



Figure 2. FTIR spectra of intrafibrillar mineralized collagen fibril in the presence of different sequestration analogs. 101x81mm (300 x 300 DPI)


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Figure 3. (A) TGA curves (B) Replotted TGA curves using dry-weight (150 °C) as reference; (C) mass ratio of mineral and collagen of intrafibrillar mineralized collagen fibrils in the presence of different sequestration analogs. 135x38mm (300 x 300 DPI)



Figure 4. Bright field (A-C) and HAADF TEM (D-F) images of freeze-dried fibrils using PAA (A/B) or PEGCOOH (C/D), and PEG-PAA (E/F) as sequestration analog. The inset in C suggests that the minerals deposited are either nanocrystalline or amorphous. 101x70mm (300 x 300 DPI)


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Figure 5. TEM (A-C), HRTEM (D) images and EDS mapping (E-H) of ACP nano-precursor formed in the presence of PEG-PAA copolymer (CP-1). 127x65mm (300 x 300 DPI)



Figure 6. Schematic illustration of the intrafibrillar mineralization mechanism of collagen fibrils induced by brush-like polymers. 101x80mm (300 x 300 DPI)


Page 32 of 32

Enhanced Intrafibrillar Mineralization of Collagen Fibrils Induced by

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