In vertebrates, the triple helical structure (TH) is a major structural pattern observed in collagen , consisting in three supercoloidal ppII conformed by amino acid residues arranged in repeated sequences X-Y-Gly, where X, Y are very frequently L-proline (Pro) and 4R-hydroxy- L-proline (4R-Hyp), respectively. Hydroxylation of Pro in Y position is essential to the fold and to the stabilization of TH . In each collagen chain containing near 1000 residues, more than 100 residues are 4r-Hyp . CD analysis gave information about the secondary structure of extracted collagen in solution [ [11, 12]. CD measurements (Fig. 1) showed the characteristic curve for the collagen triple helical structure [13, 14] with a minimum strong negative band close to 200 nm and a weak positive band close to 220 nm, indicating the presence of random coil structures, consistent with the spectrum of polyproline II Helices (ppII) [15, 16].
To evaluate the purification process of the extracted collagen and to verify significant changes on the molecular structure of collagen during self-assembly, we used FT-IR.
The three IR spectra from Fig. 2 represent the collagen in three different conditions: freeze-dried, dialyzed and undialyzed. There are typical signals from the composition of collagen type I, such the signal for amide A around 3400 cm− 1, in which there’s a NH stretching along with the hydrogen bonds, and the amide B signal, visible as a peak near to 2900 cm− 1, in which there is a symmetric stretching for CH2 .
The peaks for the Amides I, II and III matched the values described for other collagens. Amide I shows a peak near 1640–1670 cm− 1 owing to the stretching of the double bond between C-N and C-O . The C-O group indeed generates hydrogen bonds with adjacent chains, which explains the change of the peak for Amide A to lower wavelengths as the result of an increasing number of hydrogen bonds . The signal around 1530–1590 cm− 1 reveals the presence of Amide II , due to the bending for the N-H bond and the vibrational contributions because of the interaction between N and C . Finally, the Amide III signal occurs at 1250–1260 cm− 1 , as a results of the combination of the bending of the NH group and vibrational stretching of CN .
Furthermore, the low frequency signal for amide V can be identified, which is not observed in collagen without dialysis, close to 650 cm− 1, usually identified in a frequency range from 575 to 775 cm− 1, attributed to the wave motion of the N-H bond and mainly to the movements of the CH2 links .
There is a clear difference in the collagen without dialysis: besides an overall increase on the spectra, it presents two pronounced peaks at 1540 cm− 1 y 1410 cm− 1. This fact indicates that NaOH, necessary for crosslinking the fibers, reacts with the acetic acid, producing sodium acetate, whose representative peaks are 1560 cm− 1 and 1413 cm− 1 [24, 25], corresponding to the stress frequency of carbonyl group; which in turn is deposited into the self-assembled fibrils. Also, the band related to the CO bond close to 1090 cm− 1 is more intense in the collagen without dialysis due to the presence of COO-Na +, as well as the signal that is in 800 cm− 1 from the mineral phase and is only present in this spectrum.
Freeze-dried and dialyzed collagen show a small difference, barely noticeable on the changes of frequency, which are smaller for the dialyzed collagen in some absorption bands and can be caused by the conformational transitions in the structures . These results show that the collagen composition was not affected by the self-assembly method used. They also demonstrate that the dialysis process after self-assembly was effective.
Calorimetry curves (DSC) for freeze-dried, dialyzed and undialyzed collagen were compared to examine the thermal properties and stability of the protein. Figure 3 shows a denaturalization temperature of 85 °C for undialyzed collagen, where the triple helix is opened . Another transition temperature is observed around 60 °C, associated to the fusion of sodium acetate trihydrate (CH3COONa) . This signal is absent in the dialyzed collagen, which shows that the purification steps of dialysis eliminated the salt remains. The signal is also absent in the freeze-dried collagen, because it was not gelated and therefore never exposed to NaOH.
The denaturalization temperature is also reported in similar values: 85 °C for dialyzed collagen, and slightly lower for freeze-dried collagen, close to 75 °C .
There is a correlation between the thermal stability of collagen and the content of imino acid (proline and hydroxyproline) through hydrogen bonds  so that a higher content of imino acid of collagen is associated with a higher thermal denaturation . However the content of amino acids is the same in lyophilized and self-assembly collagen. So the slight difference in the denaturation temperature between those collagens is that the self-assembled collagen has a micro fibrillar structure composed of several highly-arranged triple helices interacting by hydrogen bonds and forming those microfibers. In contrast the lyophilized collagen has a set of triple non-ordered helices. Hence the opening of the triple helix when it is interacting by hydrogen bridges and is highly aligned and ordered, needs more energy (higher temperature) to be the opening of the helix (denaturalization temperature).
In addition, endothermic category transitions indicate that higher enthalpies are required for collagen without dialysis and freeze-dried, in comparison with the dialyzed collagen, owing to the presence of salt and moisture respectively, substances that lead to an increment of energy to remove them. In the case of water, substantial levels of this content in the fibers network can decrease linearly the denaturation temperature .
The dialyzed collagen also absorbs moisture from the environment, but in a minor amount because of the form of the samples (dry films) compared with freeze-dried collagen .
It is also important to consider that heat capacity of collagen depends on its acidity and its concentration .
The presence of sodium acetate can also be seen in the SEM images, in collagen without dialyzing shown in Fig. 4 (a). Some crystal formations can be appreciated, they are absent in the dialyzed collagen shown in Fig. 4 (b). In the dialyzed collagen, it can be hardly observed light spots, traces of salt. The fibrillar structure can be seen in both types of collagen, with one higher porosity in the dialyzed collagen, an important feature for a biomaterial .
The morphology of the self-assembled collagen fibers can be seen in detail in Fig. 5, showing images of atomic force (AFM) microscopy.
The microfibrils of collagen form a pattern with a period (D-periodic) of 65 nm. This value can be find within the characteristic measures of 64 to 67 nm  and corresponds to the displacement of each molecule in the axial direction, with respect to the adjacent molecule . It could be considered that this variation in the periodic spacing is intrinsic to the collagen type I self-assembly process, because of local changes in the mechanical stresses. Those changes are due to the variations in the intra-fibrillary interactions, including hydrophobic and electrostatic interactions, hydrogen bonds and crosslinks in hydroxylysine and hydroxyprolines . Each fibril diameter is between 450 and 550 nm, which coincides with the reported value for the tendons fibrils that are up to 1 cm long and 500 nm in diameter .
The stress-strain curves of collagen type I in Fig. 6 show a characteristic non-linear elastic behavior, response that can be attributed to the straightening of the conformations of the triple helix  and the alignment of the N - and C - terminal of the crosslinking .
Undialyzed collagen exhibits an ultimate tensile strength of 9.06 MPa, much higher than the strength obtained for the dialyzed collagen, of 2.38 MPa. This is due to salt that offers greater rigidity to the collagen that has not been purified, through chemical interactions that can generate cross-linking points. This in turn makes the molecules more rigid, since the relaxation movements are disabled, increasing the collagen tensile strength .
In this study, the self-assembly process of collagen was studied titrating a solution of acetic acid -collagen with NaOH (comments from graphics). Acid –base titrations by ITC are commonly carried out for calibration  but in this case was intended to determine the pI due to the absorption or release of heat due to the collagen assembly process. The assembly depend on pH, temperature, and protein concentration. The temperature was maintained constant during the experiment and the protein concentration used was higher than the minimal critical concentration for assembly .
Figure 7a shows the neutralizing reaction without the collagen in the cell. Figure 7b shows the heat released due to the dilution of the collagen with acetic acid as a control experiment. The dilution didn´t produce a high energy release of disassembly according to the assembly values obtained. Figure 7c and d shows the release or absorption of energy due to the increase in pH and ion concentration on the collagen and gelatin acetic acid solutions, respectively. There are observed differences in the energy release rate between the collagen and gelatin, suggesting a different assembly process. Two experiment were carried out to determine if the release or absorption of energy were mainly influenced by the agglomeration due to a salting in or salting out effect and not due to the molecular interactions involving proton release or absorption of the macromolecule leading assembly. The released energy values were small due to the addition of the NaCl or sodium acetate buffer to the collagen-acetic acid solution, suggesting that the energy release or absorption was driven for intermolecular interactions and not due to precipitation related to high concentration of ions. However, the effect in the equilibrium conditions due to their effect on the bulk water activity is still present for each ion pair. They may influence the electrostatic interactions between charged macromolecules participating in binding events, Debye − Hückel screening effects, or change of water activity .
Figure 8a shows the area under the curve in relation to moles for the base solution over the acid and salt and Fig. 8b shows the enthalpy changes due to pH increase. The release of energy maintains similar values until the pH reaches values close to the pI. The area under the curve in relation to moles for the base solution over the acid collagen solution and for the base solution over the collagen gelatin are shown in Fig. 8c and Fig. 8d respectively.
Some studies reported that the fibrillogenesis of collagen dilutions occur above pH 5, the pre-assembly of their triple helices in poorly organized structures can begin from lower pH values and high ionic strengths .
The steepest increase in the ∆H was observed at a pH value of 6.5. The negative ∆H shows an exothermic process with higher energy release values compared with the obtained for the injection of different buffers to the acetic acid solution, referring to changes for dilution and ionic strength. F. Jiang et. al. showed the pH influence on the collagen self- assembly, collagen molecules were adsorbed under hydrodynamic flow onto mica at pH values ranging from 2.5 to 10.5 and keeping constant the electrolyte concentration and next imaged by AFM. Between pH 2.5 and 3.5 they observed elongated globules no pronounced fibrillar structures were observed but between 5.5 and 9.5 the fibrillary structures were presented in the mica surface. At the isoelectric point the collagen was self-assemble with an associated enthalpy value of 33,89 mJ for 0,01035 moles of collagen and the structure was maintained at higher pH . This information is of vital importance in tissue engineering and especially in organ bio-printing 3D, which has become a technique quite used today, to set the parameters of the operation according to the endothermic transitions.
To determine if the enthalpy obtained is due to fiber self-assembly the collagen was denatured and the same process of neutralization was done. The DSC thermograms after denaturalization are shown in Fig. 3. In both cases, care was taken to use the same protein concentration and the same acid and base solution and low viscosity solutions were used for avoiding easy precipitation of the gelatin. A steeper enthalpy change is obtained at the same pH. However, for gelatin an endothermic peak was observed at the pI, showing different assembly process and gelification between the gelatin and the collagen.
The nucleation growth mechanism of collagen fibril assembly has been reported to be driven by a polymerization reaction, starting from the monomer and because of the possible presence of covalently crosslinked oligomers , with the intervention of both, hydrophobic and ionic inter-collagen interactions . Protein molecules are random coiled (denatured) in non-aqueous medium. Polar residues seek to form hydrogen bonds and therefore create nonpermanent α-helices and β-sheets .
To conform the gelatin, it is necessary to break up the secondary and higher structures of the parent protein collagen, with varying degrees of hydrolysis of the polypeptide backbone , from a random crosslinking of primary chains, locally twisted together, so the aggregation process could be driven by the hydrophobic effect .