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Invivosynthesisoftissuesandorgans
Introduction:
An idea was considered as to producing an entire organ in vivo by bypassing many of the steps
like cell isolation and expansion, culturing in bioreactors, scaffolds and growth factor delivery ect.
involved in traditional tissue engineering. This concept was called the in vivo bioreactor (IVB).
The following four criteria are deemed necessary for the IVB paradigm to be operative and viable:
1) Identifying a location (ectopic or orthotopic) in the body with an inherent source of pluri-
or multipotent cells or progenitor cells.
2) Establishing a privileged microenvironment that excludes other cell populations.
3) Presenting a single biophysical or soluble cue that can overcome stochastic biological
noise.
4) Defining a volume for the regenerative process.
At the time of the conception of the IVB, it was envisioned that a successful outcome would for
the first time present an approach for an autologous solution for organ and tissue transplantation
and addressing the core objective of tissue engineering.
In vivo bone engineering—the bone bioreactor
Bone formation proceeds during embryonic development by two distinct
pathways:
■ (1) the direction ossification by osteoblasts depositing matrix [intramembranous
ossification (IMO)] and is the pathway by which flat bones such as the facial bones and
skull are formed
■ (2) the conversion of a provisional cartilage matrix deposited by condensing MSCs by
infiltrating bone progenitor cells [endochondral ossification (EO)] and is the pathway by
which long bones are formed.
■ Surgical manipulation of periosteum is done using periosteal elevators,
■ which are chisels that are used to peal the periosteum back from the cortical bone surface.
This procedure, in addition to running the risk of damaging the bone surface and the
cambium layer, is also invasive and moreover the area below the peeled back periosteum
would be accessible to other infiltrating cells that could impede the healing response.
■ So a new surgical technique called the hydraulic elevation was developed.
■ In this novel approach, the subperiosteal space is accessed using a needle inserted at an
obtuse angle and Ringer’s solution or saline is introduced under hand-pressure using a
syringe attached to the needle and the desire area to be lifted is defined by varying the
lateral position and the penetration of the needle.
FIGURE: Creation of the IVB. Artist rendition of the periosteal hydraulic elevation procedure (A
and B) and photomicrograph showing the needle insertion step in a tibia of a New Zealand white
rabbit (C). A 25-ga need bent at an obtuse angle is inserted with the needle axis parallel to the
tibial surface and needle opening facing down, thus ensuring that the needle does not pierce
through and out of the periosteum. Through fanning motion, a desired breadth of the periosteum
can be gently liberated from the underlying cortical bone surface, thus ensuring preservation of
the pluripotent cell-rich cambium layer. IBV, In vivo bioreactor.
Now the question was what could be an ideal soluble signal to introduce to influence periosteal
cell fate?
■ In EO process, hypertrophy in chondrocytes and matrix calcification precedes bone
formation, a calcium (Ca)-rich microenvironment might tip the balance away from the
natural tendency of periosteal cells to undergo chondrogenesis and toward bone formation.
■ Calcium can have proliferative effect on cells at certain concentrations and calcium-related
signaling events are essential for proliferation and organization of endothelial cells (ECs).
■ Alginate gels that have a long history in cell encapsulation are a natural source of calcium,
as calcium is necessary to induce ionic-crosslinking to form the gel. So, in theory, an
injection of Ca-alginate should be sufficient to introduce a strong signal—calcium—within
the confined subperiosteal space.
■ Injection of Ca-alginate within the subperiosteal space in the tibia of skeletally mature
rabbits not only induced rapid proliferation of periosteal cells but also induced
angiogenesis and exclusively direct bone formation with very-to-little cartilage matrix.
To date, the in vivo bone bioreactor remains the only example of autologous bone engineering
purely through the judicious choice and placement of a biomaterial without any cell
transplantation or exogenous supplementation of growth factors.
■ It does not obey Wolff’s law which is, bone forms in response to load along lines of
mechanical stress.
In vivo cartilage engineering:
■ Periosteal cells can give rise to both osteoblasts and chondrocytes
■ Suramin is a small molecule inhibitor of the TGF-beta superfamily of proteins with a
known role in interfering with blood vessel formation. To remove calcium as a signal in
the IVB, the alginate gel was replaced with a hyaluronic acid (HA)-based gel and then
supplemented with Suramin and TGF-beta1. Within 10 days the IVB space was
reconstituted with cartilage as opposed to bone further validating the basic tenets of the
IVB paradigm.
■ Agarose was identified as the material of choice. Injection of agarose (2 w/v%) in the
subperiosteal IVB in the tibia of skeletally matured rabbits resulted in a massive cellular
response and the formation of a callus which by day 21 was made up of hypercellular
cartilage.
The IVB approach remains a singular example to date of in vivo cartilage tissue engineering
without cell implantation or growth factor supplementation and is currently being readied for
first in human studies.
■ The role of hypoxia in chondrogenesis in the IVB, gene expression levels of hypoxia
related factors: hypoxia inducible factor-1alpha and VEGF were quantified using
quantitative real-time PCR.
■ A statistically higher mRNA expression was found in tissues from IVB-injected with
agarose, thus validating the working hypothesis and rationale for choosing agarose.
■ The ability to engineer large volumes of hypercellular cartilage “on demand” can also
impact treatment of bone trauma. Ossification is the natural progression of cartilage once
it undergoes hypertrophy. Martin and co-workers have exploited this biological process in
a bone engineering approach; they have termed developmental engineering. They have
shown that cartilage engineered from marrow-derived MSCs and driven towards
hypertrophy can undergo remodeling in vivo into bone and marrow components upon
implantation in an ectopic site and using this approach they have also engineered an ectopic
marrow with a reconstitute immune system.
■ The IVB paradigm therefore, could have far-reaching impact in treating skeletal trauma.
The ability to engineer in vivo, two fundamentally different tissues: Bone—which is an
organ that is highly vascularized and innervated, and Cartilage—which is an avascular
tissue; from the same pluripotent stem cell source through judicious choice of an injectable
hydrogel was not only a first in exploiting biophysical and physicochemical attributes of
biomaterial to influence stem cell fate but was also overwhelming proof that the basic
principles of the IVB paradigm were well founded.
Induction of angiogenesis using biophysical cues—organotypic vasculature
engineering
Angiogenesis, the formation of new blood vessels, is a complex process involving orchestrated
released of trophic factors, and paracrine signaling between ECs and perivascular support cells
(pericytes). There are several pathologies that are associated with loss of vascularity and vascular
function and they include cardiac ischemia, hind limb ischemia, avascular necrosis, and diabetic
ulcers.
Pioneering efforts by Isner and Asahara in the mid-late 1990s led to the development of a clinical
strategy to induce new blood vessels (collateral vessels) in ischemic tissues from existing
vasculature using external cues, an approach they called therapeutic angiogenesis.
The idea was to inject a bolus of angiotropic factors such as VEGF or FGF to induce sprouting of
existing vasculature and recruitment of resident endothelial progenitor cells. This approach was
later expanded to include direct myocardial (tissue) gene transfer using plasmid encoding for
VEGF. One of the challenges with local delivery of proangiogenic signals is that the sprouting and
maturation of new vessels into normal or aberrant (angiomas) depends on local growth factor
concentration which is very difficult to modulate and control. ECs also lose their quiescence
following injury and get primed toward external proangiogenic cues.
Maintenance (stabilization) of neovasculature is necessary for restoring vascular homeostasis, and
in this regard, perivascular cells, that is, pericytes and vascular smooth muscle cells play a critical
role in vivo in not only providing scaffolding but also paracrine signaling necessary for blood
vessel sprouting and maturation.
Hydrogels are highly suitable for implementing mechanobiology paradigms as their mechanical
properties can be tuned on demand by altering the characteristics of the crosslinks. Carboxylated
agarose (CA) represents a new family of polysaccharides that yields physically crosslinked,
injectable hydrogels whose modulus can be tuned from 10s of Pa to 105 Pa independently of
concentration. Using CA hydrogels of varying stiffness modified with the integrin binding peptide
sequence arginine glycineaspartic acid (RGD) and supplemented with angiocrine factors as a
screening platform, we discovered that apicalbasal polarization in ECs could be induced within
soft hydrogels with a modulus around 2050 Pa. This hydrogel stiffness incidentally corresponds
to the mechanical properties of embryonic tissue. Under these conditions, ECs migrated and
organized themselves into free standing lumens over 100 μm in height comprising 35 ECs per
lumen cross section. This organization, which is reminiscent of vasculogenesis, that is, the
formation of blood vessels from endothelial progenitor cells (which is different from sprouting
angiogenesis that is branching from existing vasculature), more importantly occurred in this gel
environment in the absence of any support cells. Since CA gels without RGD did not promote
polarization of ECs it was deemed that In Vivo Synthesis of Tissues and Organs signaling via
integrins and mechanical coupling of ECs to the gel were critical. This demonstrated that
apicalbasal polarization and organization of ECs into three dimensional structures can be induced
purely by matrix mechanics, that is, even in absence of mural support cells.
Reference:
Lanza, Robert, Robert Langer, Joseph P. Vacanti, and Anthony Atala, eds. Principles of tissue
engineering. Academic press, 2020.
***************************

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In vivo synthesis of tissues and organs

  • 1. Invivosynthesisoftissuesandorgans Introduction: An idea was considered as to producing an entire organ in vivo by bypassing many of the steps like cell isolation and expansion, culturing in bioreactors, scaffolds and growth factor delivery ect. involved in traditional tissue engineering. This concept was called the in vivo bioreactor (IVB). The following four criteria are deemed necessary for the IVB paradigm to be operative and viable: 1) Identifying a location (ectopic or orthotopic) in the body with an inherent source of pluri- or multipotent cells or progenitor cells. 2) Establishing a privileged microenvironment that excludes other cell populations. 3) Presenting a single biophysical or soluble cue that can overcome stochastic biological noise. 4) Defining a volume for the regenerative process. At the time of the conception of the IVB, it was envisioned that a successful outcome would for the first time present an approach for an autologous solution for organ and tissue transplantation and addressing the core objective of tissue engineering. In vivo bone engineering—the bone bioreactor Bone formation proceeds during embryonic development by two distinct pathways: ■ (1) the direction ossification by osteoblasts depositing matrix [intramembranous ossification (IMO)] and is the pathway by which flat bones such as the facial bones and skull are formed ■ (2) the conversion of a provisional cartilage matrix deposited by condensing MSCs by infiltrating bone progenitor cells [endochondral ossification (EO)] and is the pathway by which long bones are formed.
  • 2. ■ Surgical manipulation of periosteum is done using periosteal elevators, ■ which are chisels that are used to peal the periosteum back from the cortical bone surface. This procedure, in addition to running the risk of damaging the bone surface and the cambium layer, is also invasive and moreover the area below the peeled back periosteum would be accessible to other infiltrating cells that could impede the healing response. ■ So a new surgical technique called the hydraulic elevation was developed. ■ In this novel approach, the subperiosteal space is accessed using a needle inserted at an obtuse angle and Ringer’s solution or saline is introduced under hand-pressure using a syringe attached to the needle and the desire area to be lifted is defined by varying the lateral position and the penetration of the needle.
  • 3. FIGURE: Creation of the IVB. Artist rendition of the periosteal hydraulic elevation procedure (A and B) and photomicrograph showing the needle insertion step in a tibia of a New Zealand white rabbit (C). A 25-ga need bent at an obtuse angle is inserted with the needle axis parallel to the tibial surface and needle opening facing down, thus ensuring that the needle does not pierce through and out of the periosteum. Through fanning motion, a desired breadth of the periosteum can be gently liberated from the underlying cortical bone surface, thus ensuring preservation of the pluripotent cell-rich cambium layer. IBV, In vivo bioreactor. Now the question was what could be an ideal soluble signal to introduce to influence periosteal cell fate? ■ In EO process, hypertrophy in chondrocytes and matrix calcification precedes bone formation, a calcium (Ca)-rich microenvironment might tip the balance away from the natural tendency of periosteal cells to undergo chondrogenesis and toward bone formation. ■ Calcium can have proliferative effect on cells at certain concentrations and calcium-related signaling events are essential for proliferation and organization of endothelial cells (ECs).
  • 4. ■ Alginate gels that have a long history in cell encapsulation are a natural source of calcium, as calcium is necessary to induce ionic-crosslinking to form the gel. So, in theory, an injection of Ca-alginate should be sufficient to introduce a strong signal—calcium—within the confined subperiosteal space. ■ Injection of Ca-alginate within the subperiosteal space in the tibia of skeletally mature rabbits not only induced rapid proliferation of periosteal cells but also induced angiogenesis and exclusively direct bone formation with very-to-little cartilage matrix. To date, the in vivo bone bioreactor remains the only example of autologous bone engineering purely through the judicious choice and placement of a biomaterial without any cell transplantation or exogenous supplementation of growth factors. ■ It does not obey Wolff’s law which is, bone forms in response to load along lines of mechanical stress. In vivo cartilage engineering: ■ Periosteal cells can give rise to both osteoblasts and chondrocytes ■ Suramin is a small molecule inhibitor of the TGF-beta superfamily of proteins with a known role in interfering with blood vessel formation. To remove calcium as a signal in the IVB, the alginate gel was replaced with a hyaluronic acid (HA)-based gel and then supplemented with Suramin and TGF-beta1. Within 10 days the IVB space was reconstituted with cartilage as opposed to bone further validating the basic tenets of the IVB paradigm. ■ Agarose was identified as the material of choice. Injection of agarose (2 w/v%) in the subperiosteal IVB in the tibia of skeletally matured rabbits resulted in a massive cellular response and the formation of a callus which by day 21 was made up of hypercellular cartilage.
  • 5. The IVB approach remains a singular example to date of in vivo cartilage tissue engineering without cell implantation or growth factor supplementation and is currently being readied for first in human studies. ■ The role of hypoxia in chondrogenesis in the IVB, gene expression levels of hypoxia related factors: hypoxia inducible factor-1alpha and VEGF were quantified using quantitative real-time PCR. ■ A statistically higher mRNA expression was found in tissues from IVB-injected with agarose, thus validating the working hypothesis and rationale for choosing agarose. ■ The ability to engineer large volumes of hypercellular cartilage “on demand” can also impact treatment of bone trauma. Ossification is the natural progression of cartilage once it undergoes hypertrophy. Martin and co-workers have exploited this biological process in a bone engineering approach; they have termed developmental engineering. They have shown that cartilage engineered from marrow-derived MSCs and driven towards hypertrophy can undergo remodeling in vivo into bone and marrow components upon implantation in an ectopic site and using this approach they have also engineered an ectopic marrow with a reconstitute immune system. ■ The IVB paradigm therefore, could have far-reaching impact in treating skeletal trauma. The ability to engineer in vivo, two fundamentally different tissues: Bone—which is an organ that is highly vascularized and innervated, and Cartilage—which is an avascular tissue; from the same pluripotent stem cell source through judicious choice of an injectable hydrogel was not only a first in exploiting biophysical and physicochemical attributes of biomaterial to influence stem cell fate but was also overwhelming proof that the basic principles of the IVB paradigm were well founded. Induction of angiogenesis using biophysical cues—organotypic vasculature engineering Angiogenesis, the formation of new blood vessels, is a complex process involving orchestrated released of trophic factors, and paracrine signaling between ECs and perivascular support cells
  • 6. (pericytes). There are several pathologies that are associated with loss of vascularity and vascular function and they include cardiac ischemia, hind limb ischemia, avascular necrosis, and diabetic ulcers. Pioneering efforts by Isner and Asahara in the mid-late 1990s led to the development of a clinical strategy to induce new blood vessels (collateral vessels) in ischemic tissues from existing vasculature using external cues, an approach they called therapeutic angiogenesis. The idea was to inject a bolus of angiotropic factors such as VEGF or FGF to induce sprouting of existing vasculature and recruitment of resident endothelial progenitor cells. This approach was later expanded to include direct myocardial (tissue) gene transfer using plasmid encoding for VEGF. One of the challenges with local delivery of proangiogenic signals is that the sprouting and maturation of new vessels into normal or aberrant (angiomas) depends on local growth factor concentration which is very difficult to modulate and control. ECs also lose their quiescence following injury and get primed toward external proangiogenic cues. Maintenance (stabilization) of neovasculature is necessary for restoring vascular homeostasis, and in this regard, perivascular cells, that is, pericytes and vascular smooth muscle cells play a critical role in vivo in not only providing scaffolding but also paracrine signaling necessary for blood vessel sprouting and maturation. Hydrogels are highly suitable for implementing mechanobiology paradigms as their mechanical properties can be tuned on demand by altering the characteristics of the crosslinks. Carboxylated agarose (CA) represents a new family of polysaccharides that yields physically crosslinked, injectable hydrogels whose modulus can be tuned from 10s of Pa to 105 Pa independently of concentration. Using CA hydrogels of varying stiffness modified with the integrin binding peptide sequence arginine glycineaspartic acid (RGD) and supplemented with angiocrine factors as a screening platform, we discovered that apicalbasal polarization in ECs could be induced within soft hydrogels with a modulus around 2050 Pa. This hydrogel stiffness incidentally corresponds to the mechanical properties of embryonic tissue. Under these conditions, ECs migrated and organized themselves into free standing lumens over 100 μm in height comprising 35 ECs per
  • 7. lumen cross section. This organization, which is reminiscent of vasculogenesis, that is, the formation of blood vessels from endothelial progenitor cells (which is different from sprouting angiogenesis that is branching from existing vasculature), more importantly occurred in this gel environment in the absence of any support cells. Since CA gels without RGD did not promote polarization of ECs it was deemed that In Vivo Synthesis of Tissues and Organs signaling via integrins and mechanical coupling of ECs to the gel were critical. This demonstrated that apicalbasal polarization and organization of ECs into three dimensional structures can be induced purely by matrix mechanics, that is, even in absence of mural support cells. Reference: Lanza, Robert, Robert Langer, Joseph P. Vacanti, and Anthony Atala, eds. Principles of tissue engineering. Academic press, 2020. ***************************