The present invention provides systems and methods for purification and concentration of autologous alpha-2-macroglobulin (A2M) from whole blood. Also provided are diagnostic methods for identifying sites in the synovial joints, spine, tendons or ligaments for treatment of pain, degeneration, or inflammation with autologous A2M. Methods for utilizing autologous A2M in combination with other autologous treatments (e.g. platelets and other growth factors) are provided in addition to combinations with exogenous drugs or carriers. Also provided is a method of producing recombinant A2M wild type or variants thereof where the bait region was modified to enhance the inhibition characteristics of A2M and/or to prolong the half-life of the protein in joints and spinal disc or epidural space.

The present invention provides systems and methods for purification and concentration of autologous alpha-2 macroglobulin (A2M) from whole blood and or recombinant A2M. Also provided are methods of treating wounds with A2M. Methods for utilizing A2M in combination with other treatments (e.g., platelets and other growth factors) are provided in addition to combinations with exogenous drugs or carriers. Also provided is a method of producing recombinant A2M wild type or variants thereof where the bait region was modified to enhance the inhibition characteristics of A2M and/or to prolong the half-life of the protein for treating wounds.

The present invention provides systems and methods for purification and concentration of autologous alpha-2 macroglobulin (A2M) from whole blood and or recombinant A2M. Also provided are methods of treating wounds with A2M. Methods for utilizing A2M in combination with other treatments (e.g., platelets and other growth factors) are provided in addition to combinations with exogenous drugs or carriers. Also provided is a method of producing recombinant A2M wild type or variants thereof where the bait region was modified to enhance the inhibition characteristics of A2M and/or to prolong the half-life of the protein for treating wounds.

The present invention provides a liquid composition comprising: (a) alpha-2-macroglobulin (A2M) isolated from a biological sample from an individual mammal, wherein the A2M is present at a concentration of at least 1.1 times higher than the concentration of A2M present in the biological sample from the individual mammal; and (b) plasma, bone marrow aspirate (BMA), or another body fluid from the biological sample from the individual mammal of (a); wherein the liquid composition is non-immunogenic to the individual mammal.

The present invention provides a composition comprising an isolated recombinant A2M polypeptide, wherein the recombinant A2M polypeptide comprises one or more non-natural bait regions, wherein the one or more non-natural bait regions replaces a wild-type A2M bait region and comprises one or more protease recognition sites not present in a wild-type A2M polypeptide, and wherein the recombinant A2M polypeptide is characterized by at least a I 0°/o enhanced inhibition of two or more different proteases selected from serine proteases, threonine proteases, cysteine proteases, aspartate proteases, glutamic acid proteases, and metalloproteases, compared to wild-type A2M.

The present invention provides an apparatus comprising: a flow filtration module comprising an inlet, an outlet, and one or more filters fluidly connected between the inlet and outlet; and a centrifuge, wherein a flow of a supernatant of a biological sample from a mammal obtained by centrifuging the biological sample with the centrifuge passes through the one or more filters to produce a composition comprising an alpha-2-macroglobulin (A2M) polypeptide at a concentration that is at least 1.1 times higher than the concentration of A2M polypeptide present in the biological sample; wherein the composition further comprises plasma, bone marrow aspirate (BMA), or another body fluid from the biological sample; and wherein the one or more filters has a pore size with a molecular weight cut-off of at most 500 kDa. The invention further provides an apparatus comprising: a flow filtration module comprising an inlet, an outlet, and one or more filters fluidly connected between the inlet and outlet; and a pump in fluid connection with the filtration module, wherein the pump is coupled to the filtration module upstream of the inlet or downstream of the outlet, wherein activation of the pump produces a flow of the biological sample from the inlet to the outlet and through the one or more filters to produce a composition comprising an alpha-2-macroglobulin (A2M) polypeptide at a concentration that is at least 1.1 times higher than the concentration of A2M polypeptide present in the biological sample.

The present invention provides biomarkers, including isolated fibronectin-aggrecan complexes that correlate with spinal or joint pain and inflammation, and methods for their detection are provided. Also provided are methods for identifying treatment sites in the spine or joint for treatment of pain and inflammation by detecting the presence of, or increased levels of, fibronectin-aggrecan complexes. Methods for treating spinal or joint pain and inflammation are also provided.

The present invention provides biomarkers, including isolated fibronectin-aggrecan complexes that correlate with spinal or joint pain and inflammation, and methods for their detection are provided. Also provided are methods for identifying treatment sites in the spine or joint for treatment of pain and inflammation by detecting the presence of, or increased levels of, fibronectin-aggrecan complexes. Methods for treating spinal or joint pain and inflammation are also provided.

The present invention provides biomarkers, including isolated fibronectin-aggrecan complexes that correlate with spinal or joint pain and inflammation, and methods for their detection are provided. Also provided are methods for identifying treatment sites in the spine or joint for treatment of pain and inflammation by detecting the presence of, or increased levels of, fibronectin-aggrecan complexes. Methods for treating spinal or joint pain and inflammation are also provided.

The present invention provides methods, reagents and kits for diagnosing and/or for the prognosis of non-autoimmune acute joint inflammation by detecting cytokine biomarkers in a sample obtained from an individual thought to be suffering from joint injury. The cytokine biomarkers used with the methods and kits of the present invention are IL-6, MIP-1β, MCP1 and IFNγ.