Gelatin Enhances the Wet Mechanical Properties of Poly(D,L-Lactic Acid) Membranes
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe research is focused on preparation of tissue membranes by electrospinning from polymeric mixture consisting from poly-lactic acid and gelatin.
The main advantage of the paper is that by combining poly-lactic acid with gelatin the mechanical and chemical parameters of prepared membranes are improved. The matrix is fully biocompatible. Mechanical properties of hydrated membranes are good with respect to targeted applications in medicine.
Using FTIR technique molecular changes of the matrix based on PDLLA/gelatin proved to be via non-covalent forces. Electrospinning method is suitable method to control the properties of prepared membranes. Other techniques are used in the praxis to prepare membranes (e.g. freeze drying), but with some limitations.
The methodology is appropriate for this study. Tensile strength and enzymatic degradation (in-vitro) of the membranes is provided. Moreover, the cytotoxicity of PDLLA/gelatin membranes was studied, as well as preosteoblasts cell proliferation. I appreciate water absorption capacity results of the membranes, as a function of gelatin concentration. Regarding further characterization of prepared membranes scanning electron microscopy was applied to study the morphology of prepared membranes.
Author should provide “Conclusion” chapter. I would prefer to shortly summarize (advantages, disadvantages) its results with respect to arguments presented. Moreover, the importance of the study to the practical implementation should be mentioned. Outline of future research of the department would be welcomed.
The study cited 36 references which is sufficient amount; most of them are up-to-date. Discussion parts include comparing and contrasting presented results with the literature sources and gives the readers scientific explanation.
Some Figures needs correction with respect to formal style, e.g. Figure 1 has too big font size.
2. Page 9, lines 263-264: the sol-gel transition temperature “minus 35 oC” ? Correct it; it should be 35 oC.
3. Page 12, lines 300-301: gel strength of gelatin – unit needs to be added. “300 Bloom” is correct.
Author Response
Reviewer#1
The research is focused on preparation of tissue membranes by electrospinning from polymeric mixture consisting from poly-lactic acid and gelatin.
The main advantage of the paper is that by combining poly-lactic acid with gelatin the mechanical and chemical parameters of prepared membranes are improved. The matrix is fully biocompatible. Mechanical properties of hydrated membranes are good with respect to targeted applications in medicine.
Using FTIR technique molecular changes of the matrix based on PDLLA/gelatin proved to be via non-covalent forces. Electrospinning method is suitable method to control the properties of prepared membranes. Other techniques are used in the praxis to prepare membranes (e.g. freeze drying), but with some limitations.
The methodology is appropriate for this study. Tensile strength and enzymatic degradation (in-vitro) of the membranes is provided. Moreover, the cytotoxicity of PDLLA/gelatin membranes was studied, as well as preosteoblasts cell proliferation. I appreciate water absorption capacity results of the membranes, as a function of gelatin concentration. Regarding further characterization of prepared membranes scanning electron microscopy was applied to study the morphology of prepared membranes.
Thank you for your kind comments. We have made correction carefully according to the reviewer’s comments. Revised portion is marked in red in the paper. The main corrections in the paper and the responses to the reviewer’s comments are made.
Author should provide “Conclusion” chapter. I would prefer to shortly summarize (advantages, disadvantages) its results with respect to arguments presented. Moreover, the importance of the study to the practical implementation should be mentioned. Outline of future research of the department would be welcomed.
→ A conclusion chapter including future research was implemented.
→ 5. Conclusions
We investigated the effect of gelatin content on the wet mechanical properties of PDLLA membranes by electrospinning 5 wt% PDLLA/gelatin BP membranes with gelatin content ranging from 0 to 40 wt%. Regardless of the environment, fracture stress increased and strain decreased with increasing gelatin content. Due to the hydrophilic nature of gelatin, wet strains consistently outperform dry strains. Conversely, wet stress is always lower than dry stress due to hydrolysis-induced biodegradation. PDLLA/gelatin membranes can be used as GBR inducers regardless of gelatin content due to their suitable wet mechanical properties (4.5~8.6 MPa) and degradability (1.8~34%). In addition, excellent biocompatibilities were also verified by examining cytotoxicity, cell proliferation, and adhesion.
PDLLA is a non-piezoelectric polymer, given its chemical configuration. Nowadays, biocompatible and biomimetic materials for bone tissue engineering have emerged, promoting cell and tissue growth in vitro and in vivo [37,38]. Piezoelectric poly(L-lactic acid) (PLLA) BPs can mimic the piezoelectric properties of bone, helping bone repair by converting physiological mechanical signals into electrical signals and promoting electrical potential on the surface of bone tissue. Piezoelectricity can enhance cell adhesion, proliferation, and osteogenic differentiation of bone marrow mesenchymal stem cells. Therefore, periodontal barriers based on piezoelectric materials, combined with bone grafts, may effectively enhance enhancing healing in fibrous connective tissues, bone tissues, and marrow tissues. However, further in vivo studies on piezoelectric biodegradable barrier membranes, combined with bone grafts [39] containing growth factors [40], antibiotics [36,41], and regenerative cells [42], are needed for a comprehensive clinical evaluation.
The study cited 36 references which is sufficient amount; most of them are up-to-date. Discussion parts include comparing and contrasting presented results with the literature sources and gives the readers scientific explanation.
→ The manuscript was revised according to your suggestion.(page 3;line 98~101, page 5; line 138, lin2 155, page 13; line 385~397)
Some Figures needs correction with respect to formal style, e.g. Figure 1 has too big font size.
→Thank you for your comments. The size of Fig. 1 was reduced. The figure caption in Figure 3 was corrected.
- Page 9, lines 263-264: the sol-gel transition temperature “minus 35 oC” ? Correct it; it should be 35 oC.
→Thank you for your comment. It was revised to 35 oC. - Page 12, lines 300-301: gel strength of gelatin – unit needs to be added. “300 Bloom” is correct.
→ Thank you for your comment. It was corrected.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThe topic addressed by the authors is interesting and concerns the manufacture of biodegradable materials with medical needs. The researchers produced membranes based on gelatin and lactic polyacid stabilized in hexafluoro propanol solvent.
After reviewing the article, I found a couple of comments that need to be corrected.
Abstract
A doctor, chemist, etc. specialist will not have a problem with abbreviations, but if the article will be read by a physicist, biologist then abbreviations such as HFIP or PDLLA may cause difficulties. So, please develop these abbreviations in the abstract and pay attention so that each newly - appearing abbreviation is explained.
Introduction
So what is the solvent used by you behind the abbreviation in the abstract HFIP ? is it not a derivative: 1,1,1,3,3,3-Hexafluoro-2-propanol ? The information should be given here, not later. (Line 43rd)
Results and Discussion
Is the viscosity data specified by the manufacturer or determined by the authors? If by the authors then please provide the device, parameters etc.(Line 61st)
What PDLLA concentrations of membranes are included in the chart? no legend. (Line 71st)
On the strength chart, it is not clear which curve means what (*red, black - no legend) (Line 121st)
In the case of compounds from the PLA group, we can talk more about ester groupings occurring at a wave number of 1754cm-1, rather than those originating from carboxylic acid. (Line 127th)
Can we talk about hydrolysis at this point or just degradation ? it is a process that takes place in an alkaline, acidic, enzymatic environment etc.. (Line 146th)
Information regarding the adhesion of membranes to the implanted membrane was lacking. Which wet or dry systems manifest more favorable adhesion and are less likely to fracture during operation. (Line 219th)
Please provide chemistry - what interactions, bonds are responsible in membrane structures for the formation of the so-called "gelatinous network". (Line 262nd)
Technical parameters of the device, manufacturer, country of origin, etc. (Line 321st)
No description of enzymatic degradation, what enzymes were used (only Lysosyme), with what activity etc.? (Line 338th)
No information regarding the use of a laminar chamber during microbial cell proliferation studies, was it not used? (Line 355th)
If the technical data including the manufacturer to chemical compounds like formaldehyde, ethanol, buffers, etc. were not given before, please add it and check the missing data in the whole methodology. (Line 366th)
The article lacks a summary - conclusions, an extremely important point. (Line 368th)
Comments for author File: Comments.pdf
The grammar of the language is not questionable
Author Response
Reviewer#2
The topic addressed by the authors is interesting and concerns the manufacture of biodegradable materials with medical needs. The researchers produced membranes based on gelatin and lactic polyacid stabilized in hexafluoro propanol solvent.
After reviewing the article, I found a couple of comments that need to be corrected.
Your helpful comments are highly appreciated. We revised our manuscript in response to the all comments of the reviewer. Your valuable comments will improve the clarity of our research article.
Abstract
A doctor, chemist, etc. specialist will not have a problem with abbreviations, but if the article will be read by a physicist, biologist then abbreviations such as HFIP or PDLLA may cause difficulties. So, please develop these abbreviations in the abstract and pay attention so that each newly - appearing abbreviation is explained.
→ Thank you for your comment. The full name of HFIP and PDLLA were given in abstract to improve the clarity of the article.
Introduction
So what is the solvent used by you behind the abbreviation in the abstract HFIP ? is it not a derivative: 1,1,1,3,3,3-Hexafluoro-2-propanol ? The information should be given here, not later. (Line 43rd)
→ Yes, HFIP solvent was used throughout the experiment. HFIP stands for 1,1,1,3,3,3-Hexafluoro-2-propanol according to IUPAC nomenclature.
Results and Discussion
Is the viscosity data specified by the manufacturer or determined by the authors? If by the authors then please provide the device, parameters etc.(Line 61st)
→ Viscosity data were measured by the authors. Detailed experimental procedure is described in the Materials and Methods section. (page 12; line 327~328).
→ The viscosity of the gelatin/HFIP solution was measured at 25 oC using a viscometer (DV 1M; Brookfield, Middleboro, MA, USA) with spindle No. SC4-31 at 30 rpm.
What PDLLA concentrations of membranes are included in the chart? no legend. (Line 71st)
→ The x-axis in Figure 1 represents PDLLA concentrations ranging from 3 to 9 wt%.
On the strength chart, it is not clear which curve means what (*red, black – no legend) (Line 121st)
→ Thank you for your comment. Figure caption of Figure 3 was revised.
→ Note that solid and open circles represent tensile stress and WUC.
In the case of compounds from the PLA group, we can talk more about ester groupings occurring at a wave number of 1754cm-1, rather than those originating from carboxylic acid. (Line 127th)
→ According to the references, a main FTIR peak located at 1754 cm-1 represents the stretching mode of the carboxyl groups. Carboxyl groups are a combination of two functional groups attached to a single carbon atom, namely, hydroxyl (single-bonded OH) and carbonyl (double bonded O) groups. The carboxyl (COOH) group is so-named because of the carbonyl group (C=O) and a hydroxyl group.
→ Ester, which is R-CO-OR, is the functional group for esters. The ester structural formula is, R-CO-OR, where, R, is an alkyl group, the, C=O, is the carbonyl group, and, -OR, is the alkoxy group. The ester was observed at 1243 cm-1.
→ PDLLA: racemic mixture of PDLA and PLLA enantiomers with a chiral structure containing 4 ligands. The center of chiral structure contains 4 ligands: a methyl group, a carboxyl group, a hydroxyl group and a hydrogen (see figure).
→ In this study, it is better to stick to chiral structures that contain carboxyl groups.
[ From Materials Research. 2014; 17(Suppl. 1); 33-38 ]
Can we talk about hydrolysis at this point or just degradation ? it is a process that takes place in an alkaline, acidic, enzymatic environment etc.. (Line 146th)
→ This is a degradation process. When the membrane is exposed to the human body, it is broken down by water molecules and physiological substances in body fluids. Once decomposed, it is excreted through respiration. It is revised.
→ page 5; line 145~149: BP PDLLA generally decomposes in two stages when exposed to the human body [1]. Initially, water molecules hydrolyze the long polymer chains into shorter fragments via ester bond cleavage. These fragments are then metabolized to produce carbon dioxide and water, which are eventually excreted through respiration [1].
Information regarding the adhesion of membranes to the implanted membrane was lacking. Which wet or dry systems manifest more favorable adhesion and are less likely to fracture during operation. (Line 219th)
→ Wet systems are better for adhesion. Dentists use the periodontal barrier after sufficiently wetting it before operation. The saliva in the mouth causes the barrier to become wet. It cannot be used clinically if it is too hydrophobic (dry state).
→ Wet systems (e=2.7-3.9) are less likely to fracture during operation than dry systems (e=0.35-3.0) due to better wet deformation against breakage. This may be due to the hydrophilic nature of gelatin because it can hold large amounts of water within the network without collapsing the structure.
Please provide chemistry – what interactions, bonds are responsible in membrane structures for the formation of the so-called “gelatinous network”. (Line 262nd)
→ The text has been modified as follows:
→ Gelatin exists as polypeptide chains held together by hydrogen bonds between the amino acids in adjacent chains. The semi-rigid material was then created by a liquid phase dispersed through a solid network. The helical junction regions act as bridges and allowing a fine gelatinous network to form [17,18]. (Line 267~270)
Technical parameters of the device, manufacturer, country of origin, etc. (Line 321st)
→ Thank you for your comments. Technical parameters of the devices are implemented.
→ The viscosity of the gelatin/HFIP solution was measured at 25 oC using a viscometer (DV 1M, Brookfield, Middleboro, MA, USA) with spindle No. SC4-31 at 30 rpm [7,10,13,14,32]. The chemical bonding of the PDLLA/gelatin polymers was studied using Fourier transform infrared spectroscopy (Spectrum Two, PerkinElmer, Beaconsfield, UK). Measurements were taken in absorption mode at 2 cm-1 intervals and within the wavelength range of 4000–400 cm-1, as previously described [7,10,13,14]. (Line 330~335)
No description of enzymatic degradation, what enzymes were used (only Lysosyme), with what activity etc.? (Line 338th)
→ Previously, degradation was examined by immersing the samples in a phosphate buffered solution for a long period of time. In the present study, lysozyme enzyme was used only to accelerate the degradation rate.
No information regarding the use of a laminar chamber during microbial cell proliferation studies, was it not used? (Line 355th)
→ The experiment was performed by J.H.R, Craniofacial Deformity Research Institute, College of Dentistry, Yonsei University. A laminar flow chamber was not used, but a CO2 incubator was used.
If the technical data including the manufacturer to chemical compounds like formaldehyde, ethanol, buffers, etc. were not given before, please add it and check the missing data in the whole methodology. (Line 366th)
→ Thank you for your comments. The manuscript was revised.
→ Cells attached to the membrane surface were fixed using 4% formaldehyde (Daejung Chemicals & Metals, Cheongju, Chungbuk, Republic of Korea) in PBS for 20 min at RT and then with methanol (Samchun Chemicals, Seoul, Republic of Korea) for 5 min at -20 oC [8]. PBS 1X is prepared by mixing 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4, purchased from Samchun Chemicals, in distilled water. To assess cell adhesion, PLLA/gelatin membrane morphology was examined using SEM. (Line 376~381)
The article lacks a summary - conclusions, an extremely important point. (Line 368th)
→ A conclusion chapter including future research was implemented.
→ 5. Conclusions
We investigated the effect of gelatin content on the wet mechanical properties of PDLLA membranes by electrospinning 5 wt% PDLLA/gelatin BP membranes with gelatin content ranging from 0 to 40 wt%. Regardless of the environment, fracture stress increased and strain decreased with increasing gelatin content. Due to the hydrophilic nature of gelatin, wet strains consistently outperform dry strains. Conversely, wet stress is always lower than dry stress due to hydrolysis-induced biodegradation. PDLLA/gelatin membranes can be used as GBR inducers regardless of gelatin content due to their suitable wet mechanical properties (4.5~8.6 MPa) and degradability (1.8~34%). In addition, excellent biocompatibilities were also verified by examining cytotoxicity, cell proliferation, and adhesion.
PDLLA is a non-piezoelectric polymer, given its chemical configuration. Nowadays, biocompatible and biomimetic materials for bone tissue engineering have emerged, promoting cell and tissue growth in vitro and in vivo [37,38]. Piezoelectric poly(L-lactic acid) (PLLA) BPs can mimic the piezoelectric properties of bone, helping bone repair by converting physiological mechanical signals into electrical signals and promoting electrical potential on the surface of bone tissue. Piezoelectricity can enhance cell adhesion, proliferation, and osteogenic differentiation of bone marrow mesenchymal stem cells. Therefore, periodontal barriers based on piezoelectric materials, combined with bone grafts, may effectively enhance enhancing healing in fibrous connective tissues, bone tissues, and marrow tissues. However, further in vivo studies on piezoelectric biodegradable barrier membranes, combined with bone grafts [39] containing growth factors [40], antibiotics [36,41], and regenerative cells [42], are needed for a comprehensive clinical evaluation.
Author Response File: Author Response.pdf
