A facile strategy for tuning the density of surface-grafted biomolecules for melt extrusion-based additive manufacturing applications

Melt extrusion-based additive manufacturing (ME-AM) is a promising technique to fabricate porous scaffolds for tissue engineering applications. However, most synthetic semicrystalline polymers do not possess the intrinsic biological activity required to control cell fate. Grafting of biomolecules on polymeric surfaces of AM scaffolds enhances the bioactivity of a construct; however, there are limited strategies available to control the surface density. Here, we report a strategy to tune the surface density of bioactive groups by blending a low molecular weight poly(ε-caprolactone)5k (PCL5k) containing orthogonally reactive azide groups with an unfunctionalized high molecular weight PCL75k at different ratios. Stable porous three-dimensional (3D) scaffolds were then fabricated using a high weight percentage (75 wt.%) of the low molecular weight PCL5k. As a proof-of-concept test, we prepared films of three different mass ratios of low and high molecular weight polymers with a thermopress and reacted with an alkynated fluorescent model compound on the surface, yielding a density of 201–561 pmol/cm2. Subsequently, a bone morphogenetic protein 2 (BMP-2)-derived peptide was grafted onto the films comprising different blend compositions, and the effect of peptide surface density on the osteogenic differentiation of human mesenchymal stromal cells (hMSCs) was assessed. After two weeks of culturing in a basic medium, cells expressed higher levels of BMP receptor II (BMPRII) on films with the conjugated peptide. In addition, we found that alkaline phosphatase activity was only significantly enhanced on films containing the highest peptide density (i.e., 561 pmol/cm2), indicating the importance of the surface density. Taken together, these results emphasize that the density of surface peptides on cell differentiation must be considered at the cell-material interface. Moreover, we have presented a viable strategy for ME-AM community that desires to tune the bulk and surface functionality via blending of (modified) polymers. Furthermore, the use of alkyne–azide “click” chemistry enables spatial control over bioconjugation of many tissue-specific moieties, making this approach a versatile strategy for tissue engineering applications. Graphic abstract Supplementary Information The online version contains supplementary material available at 10.1007/s42242-024-00286-2.


Nuclear magnetic resonance (NMR) spectroscopy:
All samples were dissolved in deuterated chloroform. 1 H NMR and heteronuclear multiple bond correlation (HMBC) spectra were recorded at 299.7 K using a Bruker Ascend 700 MHz NMR spectrometer and analyzed with TopSpin 4.0 software (Bruker, Germany).

Gel permeation chromatography (GPC):
Molecular weights and dispersities were determined by GPC using a Prominence-I LC-2050C3D LC (Shimadzu) system comprising of an autosampler, a Shim-pack GPC 800P guard (4.6 x 10 mm) column, followed by a Shim-pack 80M (8.0 x 300 mm) column, a refractive index detector, and a photodiode array detector.Tetrahydrofuran (THF) was used as mobile phase at a flow rate of 1.0 ml.min -1 at 40 °C.Polystyrene standards were used for calibration.
Samples were dissolved at 1 mg.mL -1 in THF, filtered, and 50 µL was injected for analysis.

Differential scanning calorimetry (DSC):
Thermal properties of homopolymers, copolymers, and blends were determined using a TA instrument DSC250.Samples (~5.0 mg) were placed in aluminum pans and, under a N2 flow, heated from 30 o C to 100 o C at a rate of 10 o C.min -1 .The samples were held isothermal for 5 min to erase thermal history.sPCL5k, sPCLM5k, poly(αClεCL-co-εCL)5k, poly(αN3εCLco-εCL)5k were cooled to -80 o C at -1.5 o C.min -1 .All used blend ratios were subjected to a cooling rate of -5 o C.min -1 .After keeping the samples isothermal for 1 min, they were heated again to 100 o C at 5 o C.min -1 .The thermal properties were determined from the cooling and second heating cycle.
Table S1.Synthesis poly(αClεCL-co-εCL) via a ring-opening polymerization of αClεCL and εCL using benzylic alcohol as initiator and Sn(Oct)2 as catalyst.εCL was dried over CaH2, and stored over molecular sieves (3Å).Toluene was dried for 24 h over molecular sieves (3Å).e) The Mn of purified products was determined by Gel Permeation Chromatography (GPC) using THF as eluent.
Retention curves can be found in figure S3.

Figure S2
. DSC heating and cooling thermograms of (A) p(αClεCL-co-εCL) and p(αN3εCLco-εCL) and (B) sPCL and sPCLM.Samples were heated to 100 °C, and kept isothermal for 5.0 min to remove thermal history.Then, as depicted in the graphs, the samples were cooled at -1.5 °C.min - and heated at 5 °C.min - .The thermal properties were determined from the cooling and second heating cycle.

Further results and discussion on the poly(αClεCL-co-εCL) synthesis
To synthesize the poly(αClεCL-co-εCL), we performed a ring opening copolymerization reaction of ε-CL and αClεCL in a 90 to 10 mol% feed ratio using benzylic alcohol as initiator.We initially set out to yield a high molecular weight polymer with an estimated molecular weight of 58.8 kg.mol -1 .However, when we used the methylene protons adjacent to hydroxyl end groups, the molecular weight was drastically lower (Table S1).
Simultaneously, we did observe full monomer conversion in 25 h.These results implied the presence of water in the reaction mixture.
In earlier work, we polymerized only εCL using Sn(Oct)2 as catalyst, and the expected molecular weight was approached [1].Therefore, we investigated if the αClεCL was the source of water.We performed a homopolymerization of αClεCL using the same reaction conditions as the copolymerization.According to structural analysis of NMR, we successfully obtained the homopolymer poly(αClεCL).Based on integration ratio of end-group versus backbone, we found a molecular weight of ~12 kg.mol -1 (Figure S8), which was roughly 4-5 times lower than expected.Next, we attempted more rigorous drying steps of the αClεCL (Table S1).All methods did not significantly change the molecular weight (Figure S3 & Table S1).Due to process of elimination, we concluded that the catalyst was the most likely source of the water.
Nevertheless, we obtained the desired molecular weight of ~5 kg.mol -1 for this study.
To demonstrate control over the reaction with the current set of chemicals, we added half the amount of initiator (0.5 equiv.), and observed an approximate doubling of the molecular weight, from 6.7 to 11.3 kg.mol -1 (Figure S10).In future studies, we recommend to consider switching to Zinc-based catalysts such as zinc bisamide as they are generally easier to dry.This type of catalyst was already used in a successful copolymerization of αClεCL and εCL [2].

Figure S14
. 1 H NMR spectra (700 MHz, CDCl3) of purified star-PCLM after varying the stoichiometric excess of the reagents relative to the hydroxyl end group.A higher excess was added, when moving towards the bottom spectrum.The degree of substitution (DoS) was determined by setting the integral of the A protons to 2.00, and subsequent calculation of IH / (IG+IH) *100.For 90% DoS, the IG triplet was distorted.Based on the integration values of the backbone protons, it was too high.Thus, here we assumed IH / IA *100 for the DoS.Table S2.Melting transitions and of melting enthalpy of polymers and blends.

b)
Crystallization temperatures were determined at a cooling rate of 5.0 o C.min -1 c) Melting transitions from the second heating run at a rate of 5.0 o C.min -1 .

d)
A bimodal melting peak was observed.
a) The stoichiometry of Sn(Oct)2 : benzylic alcohol : αClεCL : εCL was kept constant at 0.125 : 1.0 : 50 : 450 equivalents during the reaction.The monomer concentrations were kept constant at 0.4 M and 3.4 M, respectively.b)The theoretical maximum molecular weight of polymer was calculated based on the molar feed ratio and the molecular weight of the repeating unit (αClεCL or εCL), according to Mn, theoretical = 50*148,9 + 450*114,14 = 58.8kg.mol -1 .c) The degree of chloride incorporation in the backbone was determined via 1 H NMR. The protons adjacent to the hydroxyl groups (3.65 ppm) were set to 2.0.Then, the integral of proton adjacent to chloride (4.26 ppm) was divided by the integral of the backbone (1.38 ppm), according to IA'/(IC/2)*100.d) The Mn was determined via 1 H NMR (700 MHz, CDCl3).The integral of the methylene protons adjacent to the hydroxyl end group was set to 2.00.Then, the unit molecular weight of the εCL and αClεCL were multiplied by the normalized integral value of the backbone, according to (IE/2)*114.14 + (IA'/1)*148.9.The 1 H NMR spectra of all entries can be found in figure S5-S7.

Figure S8 .
Figure S8.A) GPC traces of poly(αClεCL-co-εCL) synthesized according to entry 1 of TableS1(red), and entry 1 using half (0.5 equiv.) the amount of initiator (blue).(B) 1 H NMR spectrum (700 MHz, CDCl3) of poly(αClεCL-co-εCL) synthesized using either (B) 1.0 or (C) 0.5 equivalents of initiator.Both GPC and NMR indicated roughly a doubling of the molecular weight when the amount of initiator was halved.

Figure S10. 1 H
Figure S10. 1 H-15 N Heteronuclear Multiple Bond Correlation spectrum (700 MHz, CDCl3) of poly(αN3εCL-co-εCL).The protons at 3.84 ppm are adjacent to two nitrogen atoms that match the expected shift of an alkyl azide.

Figure S15 .
Figure S15.Qualitative assessment of the viscosity via vial inversion tests.We first melted the polymers and then inverted the vial.0.002 wt% of hydrophobic dye and the stirrer bar were added to visualize flowing of the material.

Figure S16 .
Figure S16.DSC thermogram of the second heating cycle of (A) PCL, PCL:poly(αClεCL-co-εCL)5k, and poly(αClεCL-co-εCL)5k as well as (B) PCL, PCL:sPCL5k, and sPCL5k.Samples were heated to 100 °C, and held isothermal for 5 min to erase thermal history.Then, we cooled the samples at a rate of -5 °C.min - .In the figure, one can find the second heating cycle at 5 °C.min - .

Figure S18 .
Figure S18.Compression stress strain curves of 0-90 scaffolds.The scaffolds were printed from blends comprising several weight ratios of PCL75k to low molecular weight polymer (sPCL5k or poly(αClεCL-co-εCL)5k).Representative curves were fitted in Figure 4 of the main text.

Figure S21 .
Figure S21.(A-F) All emission curves (λexc = 550 nm) of dissolved thermopressed surfaces after incubation of an alkynated dye on different compositions.In E&F, no copper or sodium ascorbate were present.(G) Representative emission curves.N=3.