Hypoxia preconditioned plasma

Image of a prototype of the EmaCure bioreactor device for one-step preparation and sampling of Hypoxia Preconditioned Plasma (HPP)

Hypoxia preconditioned plasma or hypoxia pre-conditioned plasma (abbreviated as HPP), is the term used to describe the (cell-free) plasma obtained after extracorporeal conditioning (i.e. culturing) of anticoagulated blood under physiological temperature (37 °C) and physiological hypoxia (1–5 %O2). Blood conditioning is typically carried out over 2 to 4 days.[1] Since blood cells sediment over time, during conditioning, HPP can be passively separated from the blood cell components without centrifugation, a requirement for sampling normal blood plasma. Hypoxia precondtioned blood products (in Europe marketed as HYPPP®[1])) are currently developed as therapies for wound regeneration and skin rejuvenation.

Development

Cellular hypoxic preconditioning has been employed to obtain complex, and physiological, secretomes rich in angiogenic factors. Hypoxic preconditioning can be carried out by culturing cells in an oxygen-control incubator, or by allowing cells to create a natural hypoxic microenvironment through their own oxygen consumption. For example, skin fibroblasts that were cultured in 3D collagen matrices at high densities generated hypoxia within the matrix core, which led to an upregulation of VEGF expression.[2] Delivery of such hypoxia-induced factors in vivo has proved to be effective in supporting the vascularization, oxygenation and host-integration of subcutaneously-placed collagen implants.[3][4] Exposing peripheral blood cells to hypoxic stress stimulation has been shown to promote upregulation of expression of angiogenic growth factors (e.g. VEGF).[5] Controlled release of peripheral blood cell-derived factor mixtures was found to induce directional microvessel growth in vitro.[5] The term 'hypoxia preconditioned plasma' was first described by Hadjipanayi & Schilling, when they proposed that hypoxia can provide a useful tool for enhancing the angiogenic potential of blood plasma by generating compositions based on blood cell natural responses to a wound-like microenvironment.[6]

Preparation

The method for preparation of hypoxia preconditioned blood products employs a process through which blood is incubated (conditioned) outside the body under physiological temperature and low oxygen tension (hypoxia), i.e. under conditions resembling those encountered in vivo within a wound. A one-step bioreactor device that can be used to collect and culture (autologous) peripheral blood, then sample the protein factors present in cell-free HPP within a matrix delivery vehicle, has also been developed as a means of topical delivery of factors onto a wound.[5] Patent protection of the device design was filed in January 2013 by the same group.[7]

To date, two prototypes of the device have been produced,that allow both topical as well as injectable factor delivery.[1]

Composition and Function

By culturing the blood under conditions similar to those encountered in a normal wound micro-environment (physiological temperature and hypoxia), blood cells (e.g. peripheral blood mononuclear cells) are stimulated to upregulate a range of factor proteins that support the wound healing process.[5] Hypoxia preconditioned plasma (HPP) is rich in factors (e.g. VEGF, Angiogenin, MMPs) that promote endothelial cell migration and new vessel formation (angiogenesis).[5] Generally, the longer the duration of the conditioning phase, the higher the concentration of factors present in HPP. However, the expression of certain factors (e.g. VEGF, TSP-1) undergoes temporal changes in response to hypoxia which are not linear, due to habituation of the cellular response to stress stimulation.[5]

The secretome of peripheral blood mononuclear cells (PBMCs) has been shown to improve wound healing in mice.[8] Accordingly, it has been proposed that by delivering HPP in a spatiotemporally controlled manner (using a carrier matrix) into an area of ischaemia (i.e. reduced blood supply), such as a wound, ulcer or a burn, tissue regeneration can be enhanced through support of local angiogenesis.[5][9]

Application

HPP can be topically applied onto skin using a carrier vehicle such as a biological matrix, cream, emulsion or ointment. Alternatively, HPP can be subcutaneously or intradermally injected, by combining it with a solution of thrombin/calcium. The induced activation of the coagulation cascade results in the rapid formation of a fibrin matrix that sequesters the factors present in HPP at the site of injection, and allows their controlled release [1]

Clinical use

HPP is rich in protein factors that potently stimulate angiogenesis.[5] The intended use of HPP is hard-healing wounds, ulcers and burns where a compromise in blood supply to the wound is present.[1] Also, co-delivery of HPP with grafts (e.g. fat grafts) could improve their take and integration, by establishing early angiogenic support.

HPP is also being used as an injectable therapy, to aid skin rejuvenation.[1] The solution is injected into the (deep) dermal layer, while being combined with thrombin/calcium chloride. This results in the rapid formation of a fibrin gel-like matrix at the site of injection, through activation of coagulation. This matrix sequesters the growth factors, allowing their gradual and continuous release. The fibrin matrix also forms a temporary scaffold that supports cell migration, blood vessel ingrowth and new dermal tissue formation, in a process similar to physiological wound healing.

References

  1. 1 2 3 4 5 6 "EmaCure Wound Therapy".
  2. ↑ Cheema U, et al. (Jan 2008). "Spatially defined oxygen gradients and vascular endothelial growth factor expression in an engineered 3D cell model.". Cell Mol Life Sci. 65 (1): 177–86. doi:10.1007/s00018-007-7356-8. PMID 17994289.
  3. ↑ Hadjipanayi E, et al. (Sep 2010). "Controlling physiological angiogenesis by hypoxia-induced signaling". J Control Release. 146 (3): 309–17. doi:10.1016/j.jconrel.2010.05.037. PMID 20538024.
  4. ↑ Hadjipanayi E, et al. (Aug 2011). "First implantable device for hypoxia-mediated angiogenic induction". J Control Release. 153 (3): 217–24. doi:10.1016/j.jconrel.2011.03.029. PMID 21458514.
  5. 1 2 3 4 5 6 7 8 Hadjipanayi, E; Bauer, AT; Moog, P; Salgin, B; Kuekrek, H; Fersch, B; Hopfner, U; Meissner, T; Schlüter, A; Ninkovic, M; Machens, HG; Schilling, AF (Jul 10, 2013). "Cell-free carrier system for localized delivery of peripheral blood cell-derived engineered factor signaling: towards development of a one-step device for autologous angiogenic therapy.". Journal of Controlled Release. 169 (1-2): 91–102. doi:10.1016/j.jconrel.2013.04.008. PMID 23603614.
  6. ↑ Hadjipanayi E, Schilling AF (May 2014). "Regeneration through autologous hypoxia preconditioned plasma.". Organogenesis. 10 (2). PMID 24831225.
  7. ↑ "(WO2013113821) DEVICE-BASED METHODS FOR LOCALISED DELIVERY OF CELL-FREE CARRIERS WITH STRESS-INDUCED CELLULAR FACTORS".
  8. ↑ Mildner, M; Hacker, S; Haider, T; Gschwandtner, M; Werba, G; Barresi, C; Zimmermann, M; Golabi, B; Tschachler, E; Ankersmit, HJ (2013). "Secretome of peripheral blood mononuclear cells enhances wound healing.". PLoS ONE. 8 (3): e60103. doi:10.1371/journal.pone.0060103. PMC 3606336Freely accessible. PMID 23533667.
  9. ↑ Hadjipanayi, E; Schilling, AF (Aug 8, 2013). "Hypoxia-based strategies for angiogenic induction: The dawn of a new era for ischemia therapy and tissue regeneration.". Organogenesis. 9 (4). doi:10.4161/org.25970. PMC 3903695Freely accessible. PMID 23974216.
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