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Perfusion-based co-culture model system for bone tissue engineering

  • In this work, we report on a perfusion-based co-culture system that could be used for bone tissue engineering applications. The model system is created using a combination of Primary Human Umbilical Vein Endothelial Cells (HUVECs) and osteoblast-like Saos-2 cells encapsulated within a Gelatin Methacrylate (GelMA)-collagen hydrogel blend contained within 3D printed, perfusable constructs. The constructs contain dual channels, within a custom-built bioreactor, that were perfused with osteogenic media for up to two weeks in order to induce mineral deposition. Mineral deposition in constructs containing only HUVECs, only Saos-2 cells, or a combination thereof was quantified by microCT to determine if the combination of endothelial cells and bone-like cells increased mineral deposition. Histological and fluorescent staining was used to verify mineral deposition and cellular function both along and between the perfused channels. While there was not a quantifiable difference in the amount of mineral deposited in Saos-2 only versus Saos-2 plus HUVEC samples, the location of the deposited mineral differed dramatically between the groups and indicated that the addition of HUVECs within the GelMA matrix allowed Saos-2 cells, in diffusion limited regions of the construct, to deposit bone mineral. This work serves as a model on how to create perfusable bone tissue engineering constructs using a combination of 3D printing and cellular co-cultures.

    Citation: Stephen W. Sawyer, Kairui Zhang, Jason A. Horton, Pranav Soman. Perfusion-based co-culture model system for bone tissue engineering[J]. AIMS Bioengineering, 2020, 7(2): 91-105. doi: 10.3934/bioeng.2020009

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  • In this work, we report on a perfusion-based co-culture system that could be used for bone tissue engineering applications. The model system is created using a combination of Primary Human Umbilical Vein Endothelial Cells (HUVECs) and osteoblast-like Saos-2 cells encapsulated within a Gelatin Methacrylate (GelMA)-collagen hydrogel blend contained within 3D printed, perfusable constructs. The constructs contain dual channels, within a custom-built bioreactor, that were perfused with osteogenic media for up to two weeks in order to induce mineral deposition. Mineral deposition in constructs containing only HUVECs, only Saos-2 cells, or a combination thereof was quantified by microCT to determine if the combination of endothelial cells and bone-like cells increased mineral deposition. Histological and fluorescent staining was used to verify mineral deposition and cellular function both along and between the perfused channels. While there was not a quantifiable difference in the amount of mineral deposited in Saos-2 only versus Saos-2 plus HUVEC samples, the location of the deposited mineral differed dramatically between the groups and indicated that the addition of HUVECs within the GelMA matrix allowed Saos-2 cells, in diffusion limited regions of the construct, to deposit bone mineral. This work serves as a model on how to create perfusable bone tissue engineering constructs using a combination of 3D printing and cellular co-cultures.



    It is well known that the Herbrand-Ribet theorem is about the relation between the p-th class group of cyclotomic field Q(ζp) and the Bernoulli number.

    We introduce some notations. Let F=Q(ζp) be the cyclotomic field, and

    G=Gal(Q(ζp)/Q)={σa:1ap1}

    be the Galois group, where σa(ζp)=ζap. Let ω be the Teichmuller character of group (Z/p)×, that is, a character ω:(Z/p)×Z×p such that for aZ,(a,p)=1. Then ω(a)p1=1 and ω(a)amodp. For the group ring Zp[G], where Zp is the p-adic integer ring, the idempotents are

    εi=1p1p1a=1ωi(a)σ1a, 0ip2.

    Let A be the p-part of Cl(F), which is the class group of F. Then A=p2i=0Ai, where Ai=εiA.

    The Herbrand theorem states that if p divides the numerator of the Bernoulli number Bpi, then εiA0. In 1976, Ribet [7] proved the converse of the Herbrand's theorem. So the Herbrand-Ribet theorem is as follow.

    Theorem 1.1. Let i be an odd integer with 3ip2. If p divides the numerator of the Bernoulli number Bpi, then εiA0.

    The Herbrand theorem is obtained by the properties of the Stickelberger element and the p-adic L-function. In [8], the Herbrand-Ribet theorem for function fields was obtained. In addition, Coats and Sinnott [2] proved an analogue of Stickelberger's theorem for the K2 groups.

    Throughout this paper, inspired by the above results, we obtain respectively the K2 analogue of Herbrand-Ribet theorem and the K2 analogue of the Vandiver conjecture.

    Let S be a finite set of places of F=Q(ζp) including the archimedean ones. Let OS denote the ring of S-integers in F, i.e., the ring of all aF such that v(a)0 for each place vS. Then

    0kerdSK2FdSvSκ(v)0.

    By Quillen's localization sequence, we have the isomorphism kerdSK2(OS), which is moreover a G-isomorphism if S is stable under G (see [9, P. 271]).

    Let K2(Z[ζp]) be the K2 group of the ring of algebraic integers Z[ζp], and let C be the p-part of K2(Z[ζp]). Then we have C=p2i=0Ci, Ci=εiC.

    Lemma 2.1. There exist G-isomorphisms:

    εjA/pεj+1C/p,   0jp3.

    Proof. We note an isomorphism [4]

    μpAC/p,  (2.1)

    where G acts on μpA by the formula

    (ζx)ρ=ζρxρ,  for ζμp, ρG, xA.

    We claim that the above isomorphism is a G-isomorphism. Let S be a set of the places of Q(ζp) consisting of the archimedean ones and the finite ones above p. Let Sc denote the set of complex places. Then there is a natural exact sequence (see [9, Theorem 6.2])

    0μpCl(OS)K2OS/phS1(vSScμp)00, (2.2)

    where (μp)0 denotes the subgroup of the direct sum consisting of the elements z=(zv) such that zv=0. The map hS1 is that induced by the l-th power norm residue symbols for vSSc. Since S is stable under G, the above exact sequence is sequence of G-modules with G-homomorphisms(see [9, P. 271]). By[11, Theorem 73], C and the p-part of K2(Z[ζp,1/p]) are equal to H2ét(Z[ζp,1/p],Zp(2)). Since pZ[ζp]=(1ζp)p1, the p-part of Cl(OS) is equal to A. Moreover, the fourth term in (2.2) is 0 (see [11, Example 5]), we get that (2.1) is a G-isomorphism.

    Then we consider the following homomorphism

    δ:AμpA, xζpx.

    Here, δ is not a homomorphism of G-modules. The kernel of δ is pA, so we get an isomorphism

    δ:A/pAμpA. (2.3)

    Next we give the explicit description of δ under the Galois group action. For z:=ζpn, we have σa(z)=zω(a) (see [1, Lemma 3.3]), so there is

    σa(δx)=σa(ζp)σax=ζω(a)pσax=ω(a)δ(σa(x)).

    Therefore,

    ζpεjx=ζp(1p1p1a=1ωj(a)σ1a(x))=1p1p1a=1ω(j+1)(a)σ1a(ζpx)=εj+1(ζpx).

    Hence

    δ(εjx)=ζpεjx=εj+1(ζpx)=εj+1δ(x). (2.4)

    By (2.4), the action of idempotents εj on (2.3) leads to

    εj(A/pA)εj+1(μpA).

    Since (2.1) is a G-isomorphism, combining with the above isomorphism, we obatin

    εjA/pεj+1C/p,   0jp3

    as desired.

    Next, we give the K2 analogue of the Herbrand-Ribet theorem of the field Q(ζp) as follow.

    Theorem 2.1. Let i be even, 4ip3. Then

    Ci0p|Bp+1i.

    Proof. It is clearly that

    Ci0εiC/p0,
    Ai10εi1A/p0.

    From Lemma 2.1, we have εiC/pεi1A/p. Utilizing Theorem 1.1, we get Ci0p|Bp+1i, as required.

    However, the proof of "⇒" can also be obtained by the properties of the Stickelberger element without using Theorem 1.1 and Lemma 2.1, We sketch the proof as follow.

    Considering the Stickelberger element for the cyclotomic field Q(ζp)

    θ1=p1a=1ζ(σa,1)σ1a,

    where ζ(σ,s) is the partial zeta function, we can prove that (c2ωi(c))B2,ωi annihilates Ci, moreover, for i=4,6,,p3, B2,ωi annihilates Ci.

    We now suppose Ci0. Then B2,ωi0 (mod p). Since

    B2,ωnBn+2n+2  (mod p),

    we get

    B2,ωi=B2,ωp1iBp+1ip+1i  (mod p).

    Therefore, p|Bp+1i.

    The Vandiver's conjecture states that p does not divide the class number of Q(ζp)+, where Q(ζp)+ is the maximal real subfield of the cyclotomic field Q(ζp). Equivalently, the Vandiver's conjecture says that all the even part εiA are trivial.

    Lemma 3.1. For any irregular prime p, A2i=0, where 1i14.

    Proof. From [10] (Tables §1 Bernoulli numbers), for i=1,2,3,4,5,7, we have pB2i. So from Theorem 1.1, we have Ap2i=0,i=1,2,3,4,5,7. By the reflection theorem (see [10, Theorem 10.9])

    p-rankA2ip-rankAp2i,

    we get A2i=0.

    Let Pn denote the maximal prime factor of Bn if Bn has a prime factor. For i=6,8,9,10,11,12,13,14, from [10] (Tables §1 Bernoulli numbers) we have

    P12=691, P16=3617, P18=43867, P20=617,
    P22=593, P24=2294797, P26=657931, P28=362903.

    These primes are all less than 12,000,000. But it is well know that the Vandiver conjecture has been checked to be true for all irregular primes less than 12,000,000. So we get A2i=0 for i=6,8,9,10,11,12,13,14.

    Now we can make a K2-analogue of Vandiver's conjecture as follow.

    Conjecture 3.1. For odd i, εiC=0, where C is the p-part of K2(Z[ζp]).

    It has been proved that εp3A always vanishes (see [5]) and that if the prime p3 (mod 4), then ε(p+1)/2A is trivial (see [3,6]). Combining these results with Lemmas 2.1 and 3.1, we get the following result, which checks some cases of Conjecture 3.1.

    Theorem 3.1. For any irregular prime p, C2i+1=0 (1i14), Cp2=0 and C(p+3)/2=0 if p3 (mod 4).

    We gave the K2 analogue of Herbrand-Ribet theorem and prove the case. The K2 analogue of Vandiver's conjecture was also obtained, but this case is hard to prove. However, we just check some special circumstances of it.

    The authors are thankful for the careful reviews of referees and the editor. The first author was supported by the National Natural Science Foundation of China (No. 11901079), China Postdoctoral Science Foundation (No. 2021M700751) and the Scientific and Technological Research Program Foundation of Jilin Province (No. JJKH20190690KJ; No. 20200401085GX; No. JJKH20220091KJ). The second author was supported by the National Natural Science Foundation of China (No. 11601211).

    The authors declare that there are no conflicts of interest regarding the publication of this paper.


    Acknowledgments



    This work was supported by (IGERT) DMR-DGE-1068780. This work was also partially supported by NIH R21GM129607 awarded to Pranav Soman. We would like to thank the Syracuse University Machine Shop, Lucas D. Albrecht, and Alex B. Filip for creating the polycarbonate bioreactors and printing the ABS cages. We would also like to thank Professor Megan E. Oest from SUNY Upstate Medical University for their aid in microCT analysis and histological sectioning, and Professor Zhen Ma for the use of his fluorescence microscope.

    Conflict of interest



    The authors declare no conflict of interest.

    Author contribution



    S.W.S., K.Z. J.A.H and P.S. wrote the manuscript; S.W.S. J.A.H and P.S. conceived and designed the experiments; S.W.S. and K.Z. performed cell studies and analyzed results.

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