Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
DETAILED ACTION
Claims 2-3 are cancelled. Claims 1 and 4-22 are pending and under examination.
The rejection of claims 1 and 4-22 under 35 U.S.C. 112(b) is withdrawn in light of the claim amendment amending the phrase “forming a further solidified layer” to read “forming another solidified layer”.
Priority
This application is a CON of 16/641,526 filed on 2/24/2020 now abandoned, which is a 371 of PCT/EP2018/073028 filed on 8/27/2018. The effective filing date of the current application is August 27, 2018.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1, 4-5, and 7-21 remain rejected under 35 U.S.C. 103 as being unpatentable over Naseer et al. (“Surface acoustic waves induced micropatterning of cells in gelatin methacryloyl (GelMA) hydrogels”, Biofabrication, 2017, vol. 9, number 1, 015020; previously cited) in view of Wright et al. (“Patterning of particulate films using Faraday waves”, Review of Scientific Instruments, 2003, Vol. 74, No. 9, pp.4063-4070; previously cited), Yoo (WO 2010/030964, published on March 18, 2010; previously cited), and Son et al. (“Freestanding stacked mesh-like hydrogel sheets enable the creation of complex macroscale cellular scaffolds”, Biotechnology Journal, 2016, Volume 11, Issue 4, pp.585-591; previously cited). The rejection of claim 3 is further evidenced by Nichol et al. (“Cell-laden microengineered gelatin methacrylate hydrogels”, Biomaterials, 2010, vol 31, issue 21, pp. 5536-5544; previously cited).
Regarding claim 1, Naseer teaches that a cell mixture was suspended in a 5% GelMA solution (relevant to (a)(i) providing a suspension of particulates in a hydrogel precursor) (p.3, 2.4. Preparation of the experimental setup). Slanted-finger interdigital transducers made from lithium niobate (LiNbO3) were immobilized on the stage of a microscope (relevant to container), which was connected to a radio frequency signal generator (relevant to vibrationally coupled to the vibration generator) (Experimental Section, 2.1. SAW setup fabrication). A glass slide was placed on the LiNbO3 wafer, and a PMMA chamber was placed on top of the glass slide (relevant to container having one or more inner surface portions vibrationally coupled to one or more vibrational generators) (p.3, 2nd column – 2.4. Preparation of the experimental setup). The cell encapsulated GelMA solution was released into the PMMA chamber (relevant providing a suspension of particulates in a layer of a non-crosslinked hydrogel matrix precursor in a container) (p.3, 2nd column – 2.4. Preparation of the experimental setup).
Naseer further teaches the use of surface acoustic waves (SAW) for the rapid arrangement of cells within an extracellular matrix-based hydrogel (relevant to (a)(ii) spatially partitioning the particulates within the layer of hydrogel precursor into the particulate substructure or structure) (abstract). Naseer teaches that the SAWs were applied in a fashion to create standing SAWs on the substrate, the energy of which was subsequently transferred into the gel, creating a rapid and contactless alignment of the cells (relevant to causing standing acoustic waves in the hydrogel matrix precursor) (abstract). Naseer further teaches ultraviolet radiation induced photo-crosslinking of the cell encapsulated GelMA hydrogel (relevant to (a)(iii) allowing the non-crosslinked hydrogel matrix precursor to solidify such as to form the solidified layer of hydrogel matrix having a particulate substructure embedded therein) (abstract).
Naseer does not explicitly teach using a cross-linking agent comprising a mixture of the hydrogel precursor and a chemical crosslinking agent. However, Naseer teaches the use of the same GelMA hydrogel preparation method of Nichol. Nichol uses DPBS containing 0.5% (w/v) 2-hydroxy-1-(4-(hydroxyethoxy)phenyl)-2-methyl-1-propanone as a photoinitiator (i.e. crosslinking agent) (p. 3, Materials and Methods, Hydrogel preparation and characterization). Therefore, based on the preponderance of evidence, the GelMA pre-polymer solution of Naseer necessarily contains the same photoinitiator (relevant to a hydrogel precursor comprising a cross-linking agent comprising a mixture of the hydrogel precursor and a chemical cross-linking agent) described by Nichol.
Naseer does not teach vibration pulses at frequencies of from 10 Hz to 800Hz, or forming another solidified layer of hydrogel matrix and depositing the thus formed further solidified layer on top of the previously formed layer as in steps (b) and (c).
However, Wright teaches patterning of particulate films using Faraday waves (title). Wright teaches using frequencies ranging from 150 Hz to 350 Hz (p.4068, Results - Table 1). Wright teaches that particulate patterns were observed a few seconds after a stable Faraday wave field was formed (p.4068, 2nd column end of 1st paragraph).
Yoo teaches methods of making 3-dimensional multi-layered hydrogel constructs (title). Yoo teaches that the constructs can have bioactive agents to support living cells (relevant to (b) a three-dimensional particulate structure embedded in a body formed of a hydrogel matrix) (abstract). Yoo further teaches that creating multi-layered tissue engineered composites mimics the stratified structure found in natural tissues (relevant to (c) repeating step (b) at least one, two, three or more times) (description, p. 12, paragraph [0077]).
Son teaches a method for forming large-scale cell-hydrogel assemblies via stacking cell-embedded mesh-like hydrogel sheets to create complex macroscale cellular scaffolds (abstract). Son teaches fabricating cell-embedded mesh-like hydrogel sheets containing HepG2 or NIH3T3 cells in alginate hydrogels (p.586, 1st column – 2.1. Fabrication of cell-embedded mesh-like hydrogel sheet). Son further teaches freestanding stacked hydrogel sheets were fabricated for long-term cell culturing applications using a facile stacking process where the micropatterned hydrogel sheets were aligned using a polydimethylsiloxane drainage well (relevant to (b) forming a solidified layer of a hydrogel matrix and depositing the thus formed solidified layer on top of the previously formed solidified layer of a hydrogel matrix to form a three-dimensional structure) (abstract). Son teaches that free standing stacked hydrogel sheets were formed by laminating and aligning individual hydrogel sheets, first by loading a hydrogel sheet into a PDMS drainage well, and unfolding and aligning the hydrogel sheet (relevant to (c) repeating step (b) at least one, two, three or more times) (p.586, 2nd - column 2.3. Construction of stacked hydrogel sheets). Son further teaches three to five additional hydrogel sheets were loaded into the drainage well in the same manner (p.586, 2nd - column 2.3. Construction of stacked hydrogel sheets). Son teaches that an in vitro macroscale model of complex tissue was constructed to provide precisely fabricated, controlled microenvironments that inhibited cell necrosis, with potential applications in long-term 3D macroscopic culturing and can be used to provide in vivo-like environments (p.590-591, 4. Concluding remarks).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the hydrogel formation method of Naseer to use frequencies between 150 and 350 Hz taught by Wright to spatially partition the particulates, and further repeat the process 3-5 times to form separate sheets that could then be stacked as taught by Son to form a three-dimensional multi-layer hydrogel matrix of the instant application. One would have been motivated to combine the multi-layered hydrogel composites taught by Son with the hydrogel formation method of Naseer because Son teaches that these macroscale models of complex tissue inhibit cell necrosis and provide in vivo-like environments. One would have an expectation of success that repeating the steps of a method in succession to create individual layers of hydrogel that were then stacked together would yield the predictable result of creating a multi-layered three-dimensional hydrogel structure.
It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to select a frequency between 150Hz and 350Hz, because the claimed range overlaps with the range taught by Wright. One of ordinary skill would have reasonably expected that using frequencies between 150Hz and 350Hz would predictably result in patterned particles because Wright teaches that frequencies in this range cause particulate patterns to occur within a few seconds.
Regarding claim 4, Naseer teaches the use of gelatin methacrylate as the hydrogel material (relevant to wherein the hydrogel matrix comprises gelatine methacrylate) (abstract).
Regarding claim 5, Naseer teaches that the particulates are a mixture of cells (relevant to wherein the particulates are cells) (p. 3, Materials and methods, 2.4. Preparation of the experimental setup).
Regarding claim 7, Naseer teaches that applying different SAW patterns can result in different spatial distributions of the cells, which causes differing particulate distributions.
Naseer does not teach wherein the particulate substructure or structure of a layer is formed to have a differing particulate distribution from a particulate substructure or structure of another layer.
However, Yoo teaches that different types of cells can be printed within a single construct, embedded in different hydrogel layers, and can also be differentially and spatially distributed in the different hydrogel layers (description, p. 11, paragraph [0075]). Yoo further teaches that natural tissues comprise many different cell types, matrix materials, and have various spatial distributions of different cell types and matrix matrices (description, p. 12, paragraph [0077]).
Son teaches the assembly of various microenvironments using hydrogel modules by co-culturing HepG2 cells with NIH3T3 fibroblasts (p.590, 1st column – 3.5 Stacked hydrogel sheets with different cell types provide a 3D co-culture environment). Son teaches three HepG2 cell-containing hydrogel sheets and two NIH3T3 cells could be co-cultured and maintain their viability for three days in the stacked hydrogel sheets, and could be considered as a module to support a variety of tissue culture environments by adjusting the ratio of different cell-embedded hydrogel sheets (p.590, 1st column - 3.5 Stacked hydrogel sheets with different cell types provide a 3D co-culture environment).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to form a layer having a differing particulate structure from another layer as taught by Yoo and Son by varying the SAW frequencies applied to the hydrogel precursor solution as taught by Naseer within the frequency range taught by Wright to arrive at the claimed invention. One would have considered it beneficial to create different particulate structures in different hydrogel layers because one could more closely mimic the differential spatial distributions of cells found in natural tissues.
Regarding claims 8 and 20, Naseer teaches that 2 million cardiac fibroblasts and cardiomyocyte cells were mixed with a GelMA pre-polymer solution, and the mixture was loaded into a rectangular-shaped assembly chamber (relevant to the suspension of particulates is a suspension of two or more different types of particulates) (p.7, second column). Naseer teaches that the spatial distribution can be varied by altering the input resonant frequency (relevant to claim 8: the particulates having differing particulate distributions within said layer) (p.8, 2nd column top paragraph; FIG. 4C). Naseer further teaches that the two different cells have a similar particulate distribution within the layer (relevant to claim 20: the two different types of particulates having similar particulate distributions within the layer) (p. 7, second column; Figure 4).
Regarding claim 9, Naseer teaches applying a 1 second pulse of SAW to the cell encapsulated GelMA pre-polymer solution (relevant to wherein the particulate substructure or structure of a layer is formed by subjecting the suspension of particulates in the layer of a hydrogel matrix precursor to a one vibration pulse) (p. 8, Figure 4).
Regarding claim 10, Naseer teaches applying SSAWs at different frequencies and evaluating the level of cardiac fibroblast cell alignment (Figure 4). Naseer teaches that variation of the spatial distance between the patterned cellular lines was influenced by the changes made to the SAWs frequencies and GelMA pre-polymer concentrations (p.8, 2nd column top paragraph; Figure 4).
Yoo teaches that it is known that natural tissues of an organism comprise many different cell types, matrix materials, and have various spatial distributions of different cell types and matrix matrices (description, p. 12, paragraph [0077]). Yoo teaches that engineering tissue replacements that reproduce similar stratifications found in naturally occurring tissue is desired (description, p. 1, paragraph [0003]).
Son teaches the assembly of various microenvironments using hydrogel modules by co-culturing HepG2 cells with NIH3T3 fibroblasts (p.590, 1st column – 3.5 Stacked hydrogel sheets with different cell types provide a 3D co-culture environment). Son teaches three HepG2 cell-containing hydrogel sheets and two NIH3T3 cells could be co-cultured and maintain their viability for three days in the stacked hydrogel sheets, and could be considered as a module to support a variety of tissue culture environments by adjusting the ratio of different cell-embedded hydrogel sheets (p.590, 1st column - 3.5 Stacked hydrogel sheets with different cell types provide a 3D co-culture environment).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modify the standing acoustic waves taught by Naseer into patterning multiple layers taught by Yoo and Son, because Naseer teaches that changing the SAW frequencies influenced the patterning of the cells, Yoo teaches that natural tissues have various spatial distributions of different cell types, and Son teaches such hydrogel modules can support a variety of tissue culture microenvironments. One of ordinary skill would have found it beneficial to do so, because patterning different cell types in different manners would more closely mimic natural tissue structure.
Regarding claim 11, Naseer does not teach wherein within at least one of the steps of forming a hydrogel matrix, the concentration of particulates is increased or decreased.
However, Yoo teaches that cells (i.e. particulates) can be strategically printed on all, some or none of the hydrogel layers (description, p. 11, paragraph [0075]). As discussed above, Yoo teaches that natural tissues comprise many different cell types and have various spatial distributions of different cell types.
Son teaches the assembly of various microenvironments using hydrogel modules by co-culturing HepG2 cells with NIH3T3 fibroblasts (p.590, 1st column – 3.5 Stacked hydrogel sheets with different cell types provide a 3D co-culture environment). Son teaches three HepG2 cell-containing hydrogel sheets and two NIH3T3 cells could be co-cultured and maintain their viability for three days in the stacked hydrogel sheets, and could be considered as a module to support a variety of tissue culture environments by adjusting the ratio of different cell-embedded hydrogel sheets (p.590, 1st column - 3.5 Stacked hydrogel sheets with different cell types provide a 3D co-culture environment).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to increase or decrease the particulate content in at least one of the hydrogel layers by including or omitting the presence of cells from the hydrogel precursor. The motivation for increasing or decreasing the concentration of particulates is discussed above.
Regarding claim 12, Naseer does not teach wherein the type of concentration of particulates is modified in between steps of forming layers of a hydrogel matrix.
However, Yoo teaches an embodiment for a neural tissue model wherein neurons, neural stem cells and astrocytes are printed/co-cultured in a single layer of hydrogel or on different layers of hydrogel in the 3D multi-layered construct (description, p. 44, Example 8). Yoo further teaches that it is known that natural tissues of an organism comprise many different cell types, matrix materials, and have various spatial distributions of different cell types and matrix matrices (description, p. 12, paragraph [0077]). Yoo teaches that engineering tissue replacements that reproduce similar stratifications found in naturally occurring tissue is desired [description, p. 1, paragraph [0003]).
Son teaches the assembly of various microenvironments using hydrogel modules by co-culturing HepG2 cells with NIH3T3 fibroblasts (p.590, 1st column – 3.5 Stacked hydrogel sheets with different cell types provide a 3D co-culture environment). Son teaches three HepG2 cell-containing hydrogel sheets and two NIH3T3 cells could be co-cultured and maintain their viability for three days in the stacked hydrogel sheets, and could be considered as a module to support a variety of tissue culture environments by adjusting the ratio of different cell-embedded hydrogel sheets (p.590, 1st column - 3.5 Stacked hydrogel sheets with different cell types provide a 3D co-culture environment).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine different types of particulates taught by Yoo and Son with the method of hydrogel formation of Naseer to arrive at the claimed invention. One would have an expectation of success that combining the known methods of Son and Naseer would yield the predictable result of varying particulate concentrations within individual layers of the hydrogel matrix.
Regarding claim 13, Naseer does not teach wherein within at least one of the steps of forming a layer of a hydrogel matrix, the type of hydrogel matrix is modified in between steps.
However, Yoo teaches that multi-layered three dimensional constructs can comprise more than one type of hydrogel (description, p. 3, paragraph [0010]). Yoo further teaches where the multi-layer construct has alternating different types of hydrogel materials, for example, one layer of collagen followed by one layer of fibrin.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine different types of hydrogel materials as taught by Yoo with the hydrogel formation method taught by Naseer to arrive at the claimed invention. One would have been motivated to create a hydrogel composite of different hydrogel materials as taught by Yoo because natural tissues are stratified into layers having various cells and cell matrices and it is desirable to engineer constructs that more closely mimic natural tissues. The teachings of Yoo suggest that alternating different hydrogel materials creates a multi-layered composite. One would have had a reasonable expectation of success in using different hydrogel materials as taught by Yoo to obtain a multi-layered composite hydrogel matrix.
Regarding claim 14, Naseer does not teach crosslinking multiple layers of hydrogel matrix.
However, Yoo teaches that applying a second layer of cross-linking agent promotes complete polymerization of the first hydrogel layer, thereby embedding and encapsulating the living cells in that layer, thereby limiting the possibility of accidentally washing away or displacing the cells on the hydrogel layer (description, p. 11, paragraph [0073]). Yoo further teaches that the top surface of the second layer of the cross-linking agent serves as the cross-linking material for the next hydrogel layer to be printed. The process was repeated to construct multiple layers of collagen and cells (description, p. 35, paragraph [0157)].
Son teaches fabricating cell-embedded mesh-like hydrogel sheets containing HepG2 or NIH3T3 cells in alginate hydrogels (p.586, 1st column – 2.1. Fabrication of cell-embedded mesh-like hydrogel sheet). Son further teaches freestanding stacked hydrogel sheets were fabricated for long-term cell culturing applications using a facile stacking process where the micropatterned hydrogel sheets were aligned using a polydimethylsiloxane drainage well (abstract). Son teaches that free standing stacked hydrogel sheets were formed by laminating and aligning individual hydrogel sheets, first by loading a hydrogel sheet into a PDMS drainage well, and unfolding and aligning the hydrogel sheet (p.586, 2nd - column 2.3. Construction of stacked hydrogel sheets).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine the layer-by-layer stacking method of Yoo and laminating method of Son with the hydrogel formation method taught by Naseer to arrive at the claimed invention. One would been motivated to do so because this would provide additional structural integrity to the hydrogel matrix and entrap the cells within each individual layer. Accordingly, cross-linking the deposited layers would have been advantageous because it would have ensured that the cells within a specific layer remained in that layer while the individual hydrogel layers remained stratified.
Regarding claim 15, the phrase “wherein said structure is obtained by a process according to claim 1” is interpreted as a product-by-process limitation. MPEP 2113 make clear that “Product-by-process claims are not limited to the manipulations of the recited steps, only the structure implied by the steps” and that “determination of patentability is based on the product itself”. Naseer teaches the arrangement of biological objects, such as cells, in a three-dimensional (3D) environment (abstract). Naseer further teaches the use of surface acoustic waves for the rapid arrangement of cells within an extracellular matrix-based hydrogel such as gelatin methacryloyl (GelMA) (abstract).
Regarding claim 16, the embodiment in which the crosslinking agent is a photoinitiator capable of being activated by a physical stimulus (i.e. UV radiation) is discussed above.
Regarding claim 17, the embodiment in which the hydrogel precursor is solidified using radiation is discussed above.
Regarding claim 18, Naseer does not teach that the hydrogel precursor is partially cross-linked using enzymes, pH change or ion concentration change.
However, Yoo teaches that the hydrogel precursor can be partially cross-linked using cross-linking ions to crosslink polymers (description, p. 15, paragraph [0091]). Yoo further teaches that these ions can be anions or cations, and crosslinking can be carried out by contacting the polymers with a nebulized droplet containing the dissolved ions (i.e. ion concentration change) (description, p. 15, paragraph [0091]).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to replace the use of cross-linking ions as taught by Yoo with the UV cross-linking method taught by Naseer to arrive at the claimed invention. The teachings of Naseer and Yoo are considered to be analogous art to the claimed invention because they are in the same field of biofabrication of hydrogels comprising cell particulates. One would have a reasonable expectation of success that substituting one known method of cross-linking for another would yield the predictable result of cross-linking a hydrogel by changing the ion concentration.
Regarding claim 19, Naseer does not teach wherein the particulate substructure or structure of a layer is formed to have an identical or similar particulate distribution as another layer.
However, Yoo teaches the printing of human fibroblast cells in the upper layer of two collagen layers (description, p. 36, paragraph [0161]). Yoo further teaches that to confirm reliability of printing, a ‘plus’ shaped pattern was printed (i.e. similar particulate distribution) (description, p. 36, paragraph [0161]).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine hydrogel layers having similar particulate distributions as taught by Yoo with the hydrogel formation method taught by Naseer to arrive at the claimed invention. One would have been motivated to have done so because controlling the spatial distribution of cells in a three-dimensional hydrogel matrix more closely mimics natural tissue structure.
Regarding claim 21, Naseer does not teach wherein the particulates are cells, and the cells are osteoblasts, fibroblasts, keratinocytes, human mesenchymal stem cells (hMSCs), chondrocytes, human umbilical vein endothelial cells (hUVECs).
However, Yoo teaches that cells useful for the making of multi-layered 3D constructs include stem cells such an embryonic stem cells and mesenchymal stem cells, fibroblasts, and keratinocytes (description, p. 17, paragraph [0099]). Yoo further teaches that the cells can be human cells such as human mesenchymal stem cells and human umbilical vein endothelial cells (hUVECs) (description, p. 18, paragraph [0101]).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine the different cell types taught by Yoo with the hydrogel formation method taught by Naseer to arrive at the claimed invention. One would have been motivated to do so because combining different cell types within a hydrogel matrix would more closely resemble the stratified structure found in natural tissues. One would have a reasonable expectation of success that doing so would result in an engineered tissue construct that more closely resembled natural tissue structure.
Claims 5, 6 and 22 remain rejected under 35 U.S.C. 103 as being unpatentable over Naseer et al. (“Surface acoustic waves induced micropatterning of cells in gelatin methacryloyl (GelMA) hydrogels”, Biofabrication, 2017, vol. 9, number 1, 015020; previously cited) in view of Wright et al. (“Patterning of particulate films using Faraday waves”, Review of Scientific Instruments, 2003, Vol. 74, No. 9, pp.4063-4070; previously cited), Yoo (WO 2010/030964, published on March 18, 2010; previously cited), and Son et al. (“Freestanding stacked mesh-like hydrogel sheets enable the creation of complex macroscale cellular scaffolds”, Biotechnology Journal, 2016, Volume 11, Issue 4, pp.585-591; previously cited) as applied to claim 1 above, and further in view of Zuo et al. (“Photo-cross-linkable methacrylated gelatin and hydroxyapatite hybrid hydrogel for modularly engineering biomimetic osteon”, ACS Applied Material & Interfaces, 2015, vol. 7, issue 19, pp. 10386-10394; previously cited).
The teachings of Naseer, Wright, Yoo and Son are discussed above.
Regarding claim 5, Naseer, Wright, Yoo and Son do not teach wherein the particulates are inorganic particulates.
However, Zuo teaches a methacrylated gelatin (GelMA) and hydroxyapatite (HA) (i.e. inorganic particulates) hydrogel composite for osteon biofabrication (abstract). Zuo teaches a GelMA-HA prepolymer solution that was formed into a hydrogel through UV exposure (p. 10387, 2.2.4. Preparation of GelMA-HA Microgel and Assembly).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the GelMA pre-polymer solution of Naseer to include the hydroxyapatite particles of Zuo, because this is a simple combination of adding one known particulate to a known hydrogel precursor. Both Naseer and Zuo are directed towards biofabricating hydrogel scaffolds to support cells. One would have a reasonable expectation of success that adding the hydroxyapatite particles of Zuo to the GelMA-cell hydrogel precursor solution of Naseer would predictably result in a hydrogel matrix embedded with inorganic particulates.
Regarding claim 6, Naseer, Wright, Yoo and Son do not teach wherein the inorganic particulates are capable of supporting bio-mineralization in an implant.
However, Zuo further teaches that hydroxyapatite has been proven to be bioactive to guide and induce bone formation (i.e. capable of supporting bio-mineralization), and therefore HA precipitated GelMA hydrogel might provide improved cell-matrix responses to enhance bone-related cell functional expression (p. 10387, Introduction first column, 3rd paragraph).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine the inclusion of hydroxyapatite particulates taught by Zuo with the hydrogel formation method of Naseer in view of Yoo and Wright, because Zuo teaches that hydroxyapatite is a bioactive molecule that guides and induces bone formation. One would have been motivated to do so because such a hydrogel matrix would more closely resemble natural tissue structure.
Regarding claim 22, Naseer, Wright, Yoo and Son do not teach wherein the inorganic particles are hydroxyapatite or calcium phosphate.
The embodiment where Zuo teaches a hydrogel matrix including hydroxyapatite is discussed above.
Response to Arguments
Applicant argues that the claimed process is patentable over the applied combination of documents for the same reasons explained in the previously-filed response and are incorporated herein (See Remarks dated 12/24/2025, p.1 [1]). Applicant argues that the newly applied Son reference shows the stacking of layers, however this does not support the Office’s obviousness position because Son does not show the partial crosslinking of the hydrogel precursor at the time of forming the layer in situ, moving the thus formed layer to the growing end of the stack and fully crosslinking newly placed layers to enhance adhesion between the stacks (See Remarks dated 12/24/25, p.11 1st paragraph). Applicant argues that in Son, the layers are formed ex situ are crosslinked in a mold using calcium chloride, removed from the mold and stacked in a well (See Remarks dated 12/24/2025, p.11 2nd paragraph). Applicant argues that in contrast to the claimed invention, the stacked layers of Son are immobilized by submerging the stack in fresh non-cross-linked hydrogel precursor and cross-linking the added hydrogel precursor to “glue” the stack together (See Remarks dated 12/24/25, p.11 3rd paragraph). Applicant argues that Son provides a stable stack but by other means, and a person of ordinary skill in the art would not have had a reason, let alone motivation, to modify the applied combination of 4 teachings to arrive at the claimed method let alone achieve the advantageous results obtained by the claimed method (See Remarks dated 12/24/25, p.11 last paragraph).
Applicant's arguments filed December 24, 2025 have been fully considered but are not persuasive. The Son reference teaches the creation of individual hydrogel layers that are created separately and then stacked after formation. Son teaches fabricating cell-embedded mesh-like hydrogel sheets containing HepG2 or NIH3T3 cells in alginate hydrogels (p.586, 1st column – 2.1. Fabrication of cell-embedded mesh-like hydrogel sheet). Son further teaches freestanding stacked hydrogel sheets were fabricated for long-term cell culturing applications using a facile stacking process where the micropatterned hydrogel sheets were aligned using a polydimethylsiloxane drainage well (abstract). Son teaches that free standing stacked hydrogel sheets were formed by laminating and aligning individual hydrogel sheets, first by loading a hydrogel sheet into a PDMS drainage well, and unfolding and aligning the hydrogel sheet (p.586, 2nd - column 2.3. Construction of stacked hydrogel sheets). Naseer teaches using ultraviolet radiation induced photocrosslinking of the cell encapsulated GelMA hydrogel (abstract). Thus, one of ordinary skill would reasonably expect that as additional layers are subsequently stacked and irradiated, the hydrogel matrix would further solidify to attach the layers together sequentially.
In response to applicant's argument that the examiner has combined an excessive number of references, reliance on a large number of references in a rejection does not, without more, weigh against the obviousness of the claimed invention. See In re Gorman, 933 F.2d 982, 18 USPQ2d 1885 (Fed. Cir. 1991). One of ordinary skill in the art would have found it obvious to combine the teachings of Naseer, Wright, Yoo and Son to obtain a construct comprising hydrogel layers formed individually, with each layer being constructed by selecting parameters of frequency, particulate distribution, and particulate substructure by altering the vibration frequence, particulate selection, and other parameters as taught by the prior art for the reasons discussed in the rejection above.
Conclusion
THIS ACTION IS MADE FINAL. Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to DEEPA MISHRA whose telephone number is (571) 272-6464. The examiner can normally be reached Monday - Friday 9:30am - 3:30pm EST.
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/DEEPA MISHRA/Examiner, Art Unit 1657
/LOUISE W HUMPHREY/Supervisory Patent Examiner, Art Unit 1657